Engineered protein complexes for binding metals and small molecules

Abstract

In alternative embodiments, provided are synthetic, or non-natural, protein heterodimers or homodimers whose protein / protein binding interactions are controlled through metal or small molecule binding, and methods of making and using them. In alternative embodiments, provided are metal-controlled or small molecule-controlled heterodimers or homodimers, wherein each dimer is fused or joined to half or a portion of a detectable entity, and the when the two dimers are joined or come in contact with each other because of each dimer's binding to a metal, the detectable entity's halves, now also joined or having come in contact with each other, only now can directly or indirectly generate or initiate a detectable signal. In alternative embodiments, provided are biosensors or microfluidic devices comprising synthetic, or non-natural, protein heterodimers or homodimers as provided herein.

Description

[0001] PATENT 5810.152599PCT / Love-J7 ENGINEERED PROTEIN COMPLEXES FOR BINDING METALS AND SMALL MOLECULES RELATED APPLICATIONS This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. (USSN) 63 / 684,478, Aug.19, 2024. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with government support under FA8650-22-C-7214 awarded by the Department of Defense. The government has certain rights in the invention. TECHNICAL FIELD This invention generally relates to protein design. In alternative embodiments, provided are synthetic, or non-natural, protein heterodimers or homodimers whose protein / protein binding interactions are controlled through metal or small molecule binding, and methods of making and using them. In alternative embodiments, provided are metal-controlled or small molecule-controlled heterodimers or homodimers, wherein each dimer is fused or joined to half or a portion of a detectable entity, and the when the two dimers are joined or come in contact with each other because of each dimer’s binding to a metal or small molecule, the detectable entity’s halves, now also joined or having come in contact with each other, only now can directly or indirectly generate or initiate a detectable signal. In alternative embodiments, provided are biosensors or microfluidic devices comprising synthetic, or non-natural, protein heterodimers or homodimers as provided herein. BACKGROUND The field of biomaterials relies on tools that facilitate the building of precise protein structures at the molecular level. To increase the repertoire of emerging biotechnological tools, natural proteins have been re-engineered to optimize activity, customize thermal stability, improve specificity, and increase propensity to self- associate or bind specifically to targeted proteins. The engineering of novel protein- PATENT 5810.152599PCT / Love-J7 protein interactions, which bind with high affinity and specificity, is an ongoing goal of protein design. Engineered proteins that form specific complexes upon the addition of exogenous compounds or agents, such as metal ions or small molecule, will likely continue to be integral elements of these efforts. Designed metal-controlled protein dimers could potentially be used to bring attached passenger proteins together in a predefined manner, affording both chemical and temporal control of passenger protein interactions. Research in this field has primarily resulted in the creation of novel metal-controlled homodimers, whereas there have been fewer examples of designed metal-controlled heterodimers, which is the primary focus of the research reported herein. A key aspect of intermolecular interactions for many homodimer proteins is correlated to the symmetric nature of some designed protein / protein interactions. This feature is exemplified in a designed protein, referred to as Pizza, which forms a trimer with three-fold symmetry. A designed single metal ion binding site, positioned at a symmetry axis, functions to dimerize two copies of the trimer in the presence of cadmium chloride. Additional engineering of this unique protein complex further illustrated the value of symmetrical intermolecular orientations and the utility of inter-facial metal ion coordination. Until recently, the only means of controlling protein-protein interactions with metal ions primarily entailed the use of metal-controlled homodimers. Homodimers have limited utility unless the goal is to bring a fused passenger protein into close proximity with just itself. For example, if one fuses passenger protein A to a metal- controlled homodimer (MC), generating MCA, and passenger protein B to the same MC homodimer, generating MCB, then a heterogeneous mixture of dimers will result upon adding metal. Assuming the passenger protein has no effect on homodimer assembly, one would obtain a mixture of dimer structures that consist of 25% MCA / MCA, 25% MCB / MCB, 25% MCA / MCB, and 25% MCB / MCA, resulting in only 50% of the complexes in which passenger protein A is brought into close proximity with passenger protein B. SUMMARY In alternative embodiments, provided are synthetic, non-natural protein heterodimers comprising a first domain protein or peptide and a second domain protein or peptide, PATENT 5810.152599PCT / Love-J7 wherein the first domain protein or peptide and the second protein or peptide are each independently fused, coupled or joined to a half, a section of or a portion of a detectable moiety or entity, and wherein both the first domain protein or peptide and the second protein or peptide are independently capable of specifically binding to or associating with a (the same) target compound or composition, and the target compound or composition comprises: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, PATENT 5810.152599PCT / Love-J7 a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, and when the first domain protein or peptide and the second domain protein or peptide each bind or are associated with to the same target compound or composition under aqueous conditions the half, section of or portion of the detectable moiety or entity on the first domain protein or peptide is brought into sufficient proximity to the half, section of or portion of the detectable moiety or entity on the second domain protein or peptide to initiate generation of, or generates, a detectable signal, or to generate a molecule that can generate a detectable signal, and when the first domain protein or peptide and the second domain protein or peptide are in aqueous solution without but not binding to or associated with the target compound or composition no detectable signal is generated, wherein optionally the first domain protein or peptide and the second domain protein or peptide each bind or are associated with to the same target compound or composition by electrostatic attraction or interaction, or by an electrostatic salt bridge, and optionally the first domain protein or peptide and the second domain protein or peptide comprise amino acids that are negatively charged in an aqueous solution and the target compound or composition is or comprises a positively charged ion, optionally a positively charged metal ion. In alternative embodiments of synthetic, non-natural protein heterodimers as provided herein: - the first domain protein or peptide and the second domain protein or peptide comprise a secondary structural configuration comprising an antiparallel intermolecular extension of β-sheet; - the first domain protein comprises a sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; PATENT 5810.152599PCT / Love-J7 - the heterodimer comprises: the pair SEQ ID NO:3 and SEQ ID NO:4; or, SEQ ID NO:3 and SEQ ID NO:5; - the first domain protein or peptide and the second protein or peptide are each independently covalently linked or joined to the half, section of or portion of the detectable moiety or entity; and / or - the detectable signal comprises a radioactive, fluorescent or bioluminescent signal. In alternative embodiments, provided are synthetic or non-natural protein complexes comprising: a synthetic, non-natural protein heterodimer as provided herein, and: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, PATENT 5810.152599PCT / Love-J7 arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19- 9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, wherein both the first domain protein or peptide and the second protein or peptide are independently specifically binding (or bound to) the target compound or composition. In alternative embodiments or the synthetic or non-natural protein complexes, the two second domain proteins gain a function when the two first domain proteins specifically bind to each other, and optionally the function comprises an enzymatic activity, a binding ability or results in a signaling or a fluorescent effect. In alternative embodiments, provided are cells comprising a synthetic or non- natural protein complex as provided herein, or a synthetic, non-natural protein heterodimer as provided herein, wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell. In alternative embodiments, provided are products of manufacture, or a kit, comprising or having contained therein a synthetic or non-natural protein complex as provided herein, or a synthetic, non-natural protein heterodimer as provided herein, or a cell as provided herein, and optionally the product of manufacture is fabricated as a microfluidic device or a biosensor. In alternative embodiments, provided are methods for detecting a target compound or a composition in an aqueous solution, comprising: (a) providing a synthetic, non-natural protein heterodimer as provided herein, wherein both the first domain protein or peptide and the second protein or peptide of the synthetic, non-natural protein heterodimer are independently capable of specifically binding to the same target compound or composition; PATENT 5810.152599PCT / Love-J7 (b) adding the synthetic, non-natural protein heterodimer of (a) to an aqueous solution; and (c) detecting and / or measuring whether, or not, a detectable signal is generated, wherein detection of a detectable signal indicates that the target compound or composition is present in the aqueous solution, and optionally the aqueous solution is a physiologic solution or saline. In alternative embodiments methods as provided herein further comprise adding to the solution a metal chelating compound, wherein optionally the metal chelating compound comprises EDTA, resulting in the chelation of a metal or metal ion bound to the synthetic, non-natural protein heterodimer, resulting in dissociation of the specific binding of the first domain protein or peptide and the second protein or peptide of the synthetic, non-natural protein heterodimer to the target compound or composition, and loss of generation of the detectable signal. In alternative embodiments, provided are recombinant or synthetic nucleic acids encoding SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In alternative embodiments, provided are expression vehicles comprising a recombinant or synthetic nucleic acid as provided herein, wherein optionally the expression vehicle is a viral particle, a recombinant virus, a baculovirus, a phage, a plasmid, a phagemid, a cosmid, a fosmid, a bacterial artificial chromosome, a viral DNA, or a P1-based artificial chromosomes. In alternative embodiments, provided are cells having contained therein, or comprising, a recombinant or synthetic nucleic acid as provided herein, or an expression vehicle as provided herein, or a synthetic, non-natural protein heterodimer as provided herein, wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell. In alternative embodiments, provided are kits comprising: a cell as provided herein, or a recombinant or synthetic nucleic acid as provided herein, or an expression vehicle as provided herein, or a synthetic, non-natural protein heterodimer as provided herein. In alternative embodiments, provided are biosensors or microfluidic devices comprising: a cell as provided herein, or a synthetic or non-natural protein complex as provided herein, or a synthetic, non-natural protein heterodimer as provided herein. PATENT 5810.152599PCT / Love-J7 In alternative embodiments, provided are uses of a synthetic, non-natural protein heterodimer of any of the preceding claims to make a product of manufacture as provided herein, wherein optionally the product of manufacture is or comprises a biomaterial, and optionally the biomaterial comprises a microfluidic device or a biosensor. In alternative embodiments, provided are synthetic, non-natural protein heterodimers as provided herein for use in making a product of manufacture, wherein optionally the product of manufacture is or comprises a biomaterial, and optionally the biomaterial comprises a microfluidic device or a biosensor. In alternative embodiments, provided are isolated or recombinant synthetic polypeptides comprising a sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, optionally further comprising a moiety or amino acid sequence capable of binding to a detectable compound, composition or moiety, and optionally further comprising added to the C terminus (GGSGGCC) (SEQ ID NO:41). In alternative embodiments, provided are gold nanoparticles comprising an isolated or recombinant synthetic polypeptide having a sequence as set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and gold particle. In alternative embodiments, provided are recombinant or synthetic nucleic acids encoding a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID PATENT 5810.152599PCT / Love-J7 NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40. In alternative embodiments, provided are expression vehicles comprising a recombinant or synthetic nucleic acid as provided herein, wherein optionally the expression vehicle is a viral particle, a recombinant virus, a baculovirus, a phage, a plasmid, a phagemid, a cosmid, a fosmid, a bacterial artificial chromosome, a viral DNA, or a P1-based artificial chromosomes. In alternative embodiments, provided are cells having contained therein, or comprising, a recombinant or synthetic nucleic acid as provided herein, or an expression vehicle as provided herein, or a synthetic polypeptide as provided herein, wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell. In alternative embodiments, provided are kits comprising: a cell as provided herein, or a recombinant or synthetic nucleic acid as provided herein, or an expression vehicle as provided herein, or a synthetic polypeptide as provided herein. In alternative embodiments, provided are a biosensor or a microfluidic device comprising: a cell as provided herein, or a recombinant or synthetic nucleic acid as provided herein, or an expression vehicle as provided herein, or a synthetic polypeptide as provided herein. In alternative embodiments, provided are a method for isolating a rare earth metal (REM) or rare earth element (REE) (optionally neodymium, lanthanum, dysprosium, gadolinium) comprising: (a) adding to a solution, optionally an aqueous solution, a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, wherein the solution comprises a rare earth metal (REM) or rare earth element (REE), and (b) isolating the peptide- REM or peptide-REE. In alternative embodiments, provided are methods for isolating: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc PATENT 5810.152599PCT / Love-J7 cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, comprising: PATENT 5810.152599PCT / Love-J7 adding to a solution, optionally an aqueous solution, a peptide or polypeptide as provided herein, wherein the peptide or polypeptide specifically binds to a target comprising: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, PATENT 5810.152599PCT / Love-J7 a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, and isolating a peptide-target or polypeptide-target complex. In alternative embodiments, provided are methods for isolating a rare earth metal (REM) or rare earth element (REE) (optionally neodymium, lanthanum, dysprosium, gadolinium) comprising: (a) adding to a solution, optionally an aqueous solution, a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, wherein the solution comprises a rare earth metal (REM) or rare earth element (REE), and (b) isolating the peptide- REM or peptide-REE. In alternative embodiments, the REE comprises a lanthanide, scandium or yttrium, or, the lanthanide selected from the group consisting of: Lanthanum (La) - Atomic Number 57; Cerium (Ce) - Atomic Number 58; Praseodymium (Pr) - Atomic Number 59 ;Neodymium (Nd) - Atomic Number 60; Promethium (Pm) - Atomic Number 61; Samarium (Sm) - Atomic Number 62; Europium (Eu) - Atomic Number 63; Gadolinium (Gd) - Atomic Number 64; Terbium (Tb) - Atomic Number 65; Dysprosium (Dy) - Atomic Number 66; Holmium (Ho) - Atomic Number 67; Erbium (Er) - Atomic Number 68; Thulium (Tm) - Atomic Number 69; Ytterbium (Yb) - Atomic Number 70; and, Lutetium (Lu) - Atomic Number 71. In alternative embodiments provided are methods for isolating a compound or composition, wherein the compound or composition comprises: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a rare earth element (REE), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), PATENT 5810.152599PCT / Love-J7 a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, the method comprising: (a) adding to a solution, optionally an aqueous solution, the compound or composition, and (b) isolating the compound or composition. In alternative embodiments, the REE or compound or composition is isolated or removed from the solution the by use of a: High-Performance Liquid Chromatography (HPLC); Reverse-Phase HPLC (RP-HPLC); Reversed-Phase Chromatography (RPC); Ion-Exchange Chromatography (IEX); Size-Exclusion PATENT 5810.152599PCT / Love-J7 Chromatography (SEC) immunoaffinity column; Chiral Chromatography or a combination thereof. In alternative embodiments provided are uses of a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for isolating a rare earth metal (REM) or rare earth element (REE) (optionally a lanthanide, or neodymium, lanthanum, dysprosium, gadolinium). In alternative embodiments provided are peptides or polypeptides having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for use in isolating a rare earth metal (REM) or rare earth element (REE) (optionally a lanthanide, or neodymium, lanthanum, dysprosium, gadolinium). In alternative embodiments the lanthanide selected from the group consisting of: Lanthanum (La) - Atomic Number 57; Cerium (Ce) - Atomic Number 58; Praseodymium (Pr) - Atomic Number 59 ;Neodymium (Nd) - Atomic Number 60; Promethium (Pm) - Atomic Number 61; Samarium (Sm) - Atomic Number 62; Europium (Eu) - Atomic Number 63; Gadolinium (Gd) - Atomic Number 64; Terbium (Tb) - Atomic Number 65; Dysprosium (Dy) - Atomic Number 66; Holmium (Ho) - Atomic Number 67; Erbium (Er) - Atomic Number 68; Thulium (Tm) - Atomic Number 69; Ytterbium (Yb) - Atomic Number 70; and, Lutetium (Lu) - Atomic Number 71. In alternative embodiments provided are uses of a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for isolating a compound or composition, wherein the compound or composition comprises: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a rare earth element (REE), PATENT 5810.152599PCT / Love-J7 a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA- 125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin. In alternative embodiments, provided are peptides or polypeptides having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for use in isolating a rare earth metal (REM) or rare earth element (REE) (optionally a lanthanide, or neodymium, lanthanum, dysprosium, gadolinium). PATENT 5810.152599PCT / Love-J7 The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes. DESCRIPTION OF DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.FIG.13 schematically illustrates the distribution of binding interactions for homodimer passenger protein fusions: when passenger proteins A (blue) and B (yellow) are fused to the same metal-controlled homodimer (green) the distribution will result in 25% A / A, 25% B / B, 25% A / B, and 25% B / A. FIG.2A-D3 schematically illustrates the potential hetero- and Homodimer associations: FIG.2A illustrates metal-binding histidine residues for engineered symmetric homodimer (MCD_C1); FIG.2B illustrates an exemplary designed heterodimer with one intact metal binding site at bottom and a designed intermolecular electrostatic interaction at top; FIG.2C-D illustrate represent unwanted homodimers that could form through self-binding of either of the heterodimer binding partners: FIG.2C illustrates R12H29H33 half (the R12, H33, H29 half of the dimer in FIG.2B), or, FIG.2D illustrates the H12E29E33 half (the E29, E33, H12 half of the dimer in FIG.2B) of the heterodimer variant. FIG.3 schematically illustrates PYMOL Model of the Putative ‘Networked’ Electrostatic Salt Bridge: the X-ray structure of the MCD_C1 homodimer was used to model an arginine residue at position 12 and verify that it is within an electrostatic bond length with the glutamic acid residues at positions 29 and 33 of the corresponding binding partner. FIG.4A-C graphically illustrate SEC MALS (size exclusion chromatography with multi-angle light scattering) Analysis of Single Heterodimer Variants and PATENT 5810.152599PCT / Love-J7 Complexes; black curves correspond to samples with no added metal, while red curves correspond to samples that contain 1 mM Zn2+; FIG.4A graphically illustrates MCHet_A by itself; FIG.4A graphically illustrates MCHet_B by itself; and FIG.4C graphically illustrates equimolar concentrations of MCHet_A and MCHet_B. FIG.5A-B graphically illustrate SEC MALS Analysis of Single Heterodimer Variants and Complexes; Red curves correspond to samples that contain 1 mM Zn2+, black curves correspond to samples with no added Zn2+: FIG.5A graphically illustrates MCHet_C by itself; and FIG.5B graphically illustrates equimolar concentrations of MCHet_A and MCHet_C. FIG.6 graphically illustrates SEC MALS measured Salt-Dependence on Heterodimer Formation: the five traces correspond to SEC MALS runs performed in increasing sodium chloride concentrations, and curves to the left indicate larger molecular entities and thus higher molecular weights. FIG.7A-B schematically illustrate crystal structures of the exemplary Heterodimer Complexes: FIG.7A schematically illustrates MCHet_A / _B; and, FIG.7B schematically illustrates MCHet_A / _C. FIG.8A-B schematically illustrate Comparison of the Interfacial Hydrophobic Contacts: FIG.8A schematically illustrates the MCHet_A / _B complex, where all four interfacial leucine residues are colored yellow; and FIG.8B schematically illustrates the MCHet_A / _C complex where the two leucine residues from MCHet_A are colored yellow (left) whereas the valine residues from MCHet_C (right) are colored green. FIG.9 illustrates an image of an 18% SDS-PAGE Gel of MCHet_A, MCHet_B, and MCHet_C: Lane 1 – molecular weight markers; Lane 2 – MCHet_B; Lane 3 – MCHet_A; Lane 4 – MCHet_C. FIG.10 graphically illustrates the thermal denaturation of MCHet_A, MCH_B, and MCHet_C. PATENT 5810.152599PCT / Love-J7 FIG.11 graphically illustrates an image of analytical Ultracentrifugation: Sedimentation Velocity of MCHet_A and MCHet_C. FIG.12A-B illustrate a Bacterial Service Display (BSD) system as provided herein and as described in detail, below: FIG.12A illustrates two exemplary REE-BPs displayed in the context of the multiple domain protein construct; and FIG.12B illustrates the exemplary BSD interacting with a bacterial membrane. FIG.13A-G schematically illustrates an exemplary Split Fluorescent- Protein Assay for Enhanced Metal Binding Detection: A) No metal present, the REE-BPs (pink and green) do not bind one another, resulting in no florescence. B) Upon the addition of metal, the REE-BPS will bind one another, bring β-strand 11 into proximity to β-strands 1 - 10, which enables mCherry to properly fold and fluoresce. L – linker sequences. FIG.14 schematically illustrates an exemplary Split mCherry Assay: when two MCDs are expressed as fusions with different portions (β-strands) of mCherry and incubated with metal, the MCDs dimerize and bring the split portions of mCherry together and induce mCherry to reform and fluoresce (pink, or darker, tube on right). The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims. Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION In alternative embodiments, provided are synthetic, or non-natural, protein heterodimers or homodimers whose protein / protein binding interactions are controlled through metal and / or small molecule binding, and methods of making and using them. In alternative embodiments, provided are metal-controlled or small molecule- controlled heterodimers or homodimers, wherein each dimer is fused or joined to half or a portion of a detectable entity, and the when the two dimers are joined or come in contact with each other because of each dimer’s binding to a metal and / or small molecule, the detectable entity’s halves, now also joined or having come in contact with each other, only now can directly or indirectly generate or initiate a detectable PATENT 5810.152599PCT / Love-J7 signal (in other words, when the halves of the detectable moiety are not sufficiently close or in contact with each other, there is no detectable signal generated). In alternative embodiments, designed metal-controlled or small molecule- controlled heterodimers or homodimers as provided herein are each (each dimer) fused, or genetically fused, to two sections or pieces (for example, two halves) of a detectable entity or moiety. In alternative embodiments the two proteins (each dimer) that make up the heterodimer or homodimer are each fused, or genetically fused, to a different section or part of a detectable entity or moiety such that when the heterodimer or homodimer halves are in close proximity to each other or contact each other because of their specific binding to a particular metal and / or small molecule, the now joined pieces or section of the detectable entity or moiety can now generate a detectable signal; thus, a detectable signal is generated when the metal and / or small molecule is in a solution with the metal-controlled heterodimers or homodimers, and the metal-controlled or small molecule-controlled heterodimers or homodimers can now act as metal and / or small molecule detection agents or moieties. In the absence of metal and / or small molecule, the two dimers of the heterodimers do not associate with each other or bind together, so the two pieces or sections of the detectable agent or moiety cannot generate a detectable signal; and upon addition (or detection) of the metal and / or small molecule the two pieces or sections of the detectable agent or moiety are driven to bind or contact one another, or at least come in sufficiently close contact with each other, to generate a detectable signal. This enables both temporal and chemical control of the self-association of the two pieces or sections of the detectable agent or moiety (that are attached to respectively halves of the heterodimer). In alternative embodiments the pieces or sections of the detectable agent or moiety are fused proteins, for example, are two enzymes, that are only activated when in proximity or the rate of catalysis is enhanced upon their close association. In alternative embodiments the two proteins are two halves of a ‘split’ enzyme or a ‘split’ fluorescent protein. Association (with each other by binding of the heterodimer or homodimer in the presence of metal and / or small molecule) leads to or results in the ‘turning on’ of catalysis for the split enzyme or emission of fluorescence by the two halves of a fluorescent protein. To stop catalysis or fluorescence, the addition of a metal and / or small molecule chelating agents such as EDTA or EGTA will break up PATENT 5810.152599PCT / Love-J7 the protein / protein association, thus leading to the termination of catalysis for the split enzyme or complete quenching of fluorescence for the split fluorescent protein. Metal-controlled or small molecule-controlled heterodimers and homodimers as provided herein can be used and reengineered to function as integral components of biosensors for the sensitive detection of atoms and molecules such as rare earth elements, or heavy toxic metals, and / or any form of molecular pollutant or toxins. In alternative embodiments biosensors built from the heterodimers (and homodimers) are integral components of microfluidic devices (for example, laboratories on a chip) or other detection technologies used for the ultrasensitive detection of atoms and molecules such as for example, detection of the following (where, in alternative embodiments, a heterodimers and homodimers as provided herein can specifically bind to at least one of the following, or by binding to one of the following each half of the heterodimer or homodimer associates with its paired half): Table 1 A small molecule or a drug, for example, a Schedule I drug, substance, or chemical including for example heroin, opium, lysergic acid diethylamide (LSD), marijuana (cannabis), 3,4-methylenedioxymethamphetamine (ecstasy), methaqualone, and peyote; or, a Schedule II drug including an opioid or a synthetic opioid such as for example; amphetamine or dextroamphetamine, methylphenidate, morphine, opium, codeine, hydrocodone, hydromorphone (Dilaudid®), methadone (Dolophine®), meperidine (Demerol®), oxycodone (OxyContin®, Percocet®), and fentanyl (Sublimaze®, Duragesic®); or, a Schedule III drug, such as for example: benzphetamine (Didrex®), phendimetrazine, ketamine, oxandrolone (Oxandrin®), Tylenol with codeine, ketamine, anabolic steroids, and testosterone; or a drug such as a methylxanthine, including for example: caffeine, aminophylline, 3-isobutyl-1- methylxanthine (IBMX), paraxanthine, pentoxifylline, theobromine, theophylline, and 7-methylxanthine (heteroxanthine). A metal, for example, a rare earth element, a heavy metal, for example: lead, cadmium, mercury, zinc, chromium, etc. Per- and Polyfluoroalkyl Substances (PFAS) Polychlorinated Biphenyls (PCBs) Formaldehyde, Paraformaldehyde or polyoxymethylene Asbestos or anthophyllite PATENT 5810.152599PCT / Love-J7 Pesticide Chemicals, for example, Glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, clopyralid, Organophosphates such as malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, Poisons and toxins used to kill vermin such as mice and rates. Petroleum-based toxins and pollutants Arsenic Selenium Chromium or chromium compounds such as trivalent chromium (Cr(III)) ion, toxins associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde. In alternative embodiments, provided is a heterodimer- or homodimer- comprising biosensor device engineered to function as an integral component of a microfluidic device or other detection technology that can be used, for example, for the ultrasensitive detection of atoms and molecules associated with human, plant, or animal diseases as well as various metabolic processes that take place within microorganisms, humans, plants, or animals. Examples of such atoms and molecules detected with devices as provided herein, to be used for ultrasensitive detection, include for example: Human or plant hormones such as steroids, estrogen and testosterone A drug, for example, a performance enhancing drug, or a small molecule or a compound such as a peptide, such as erythropoietin (EPO), or a steroid such as an anabolic steroid, and the like, Biomolecular markers associated with cancer such as Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, Chromogranin, Alpha fetoprotein (AFP), vimentin and the like. In alternative embodiments, provided are novel forms of synthetic, or non- natural, protein heterodimers or homodimers whose protein / protein binding interactions are controlled through metal or small molecule binding, as well as other ligand binding, and methods of making and using them. In alternative embodiments, use of synthetic, or non-natural, protein heterodimers or homodimers as provided herein allows the ability to precisely control protein complex formation, which will PATENT 5810.152599PCT / Love-J7 have high utility in the expanding field of biomaterials. Driving protein / protein binding through metal or small molecule binding (or binding of a molecule listed in Table 1) allows precise control of both dimer formation and dissociation, a control not available with constitutive protein complexes (proteins that form permanent complexes). In alternative embodiments, provided herein are synthetic, or non-natural, two protein binding-partners that are completely monomeric in the absence of metal or small molecule yet form a dimer (the two proteins associate into a specific complex) in the presence of a small molecule or metal such as zinc sulfate or molecule listed in Table 1. In addition to forming a heterodimer in the presence of metal, the complex also dissociates upon addition of a metal or small molecule chelating compound such as EDTA. In alternative embodiments, to promote the specificity of a metal-controlled heterodimer or homodimer as provided herein and to prevent self-association, elements of positive and negative design were incorporated. This was achieved through the incorporation of a cross-interface electrostatic interactions as well as additional modifications to the degree of hydrophobicity at the protein-protein interface. The resulting metal-controlled heterodimer binds with low micromolar affinity. Extensive biophysical characterization revealed that these synthetic, or non- natural, proteins as provided herein retain high thermal stability, and are completely monodisperse in both the monomeric and dimeric states, in other words, synthetic, or non-natural, proteins as provided herein remain monomeric without metal up to high protein concentrations. In alternative embodiments, provided and described herein are two exemplary heterodimers HetA and HetB. One application for the use of these heterodimeric proteins is that they can be expressed with any other protein in a controlled physical association, or in other words, heterodimeric proteins as provided herein are tethered (or otherwise attached or fused to) passenger proteins to generate engineered proteins that can form tight specific complexes. For this example, we will refer to the two other generic proteins a Pro1 and Pro2, which can be expressed as either N- or C- terminal fusions to HetA and HetB, resulting in Pro1-HetA and HetB-Pro2. Upon the addition of metal, such as zinc sulfate, association will occur between the two heterodimers, resulting in the formation of Pro1-HetA / HetB-Pro2 complex. This PATENT 5810.152599PCT / Love-J7 functions to bring Pro1 and Pro2 into close physical association which is necessary for them to perform a specific function. For example, Pro1 and Pro2 could be two halves of a ‘split’ enzyme or split fluorescent protein that only functions when the two halves of the enzyme are brought into close special proximity. This complex can be easily disassociated upon the addition of metal chelating chemicals such as EDTA. This affords an unprecedented level of chemical and temporal control over this protein complex formation. Thus, the synthetic non-natural heterodimeric proteins as provided herein act as a biomolecular on / off switch that will be used to drive two attached proteins into close proximity, but also have the ability to separate them. Products of manufacture and Kits Provided are products of manufacture and kits for using the synthetic non- natural heterodimeric proteins as provided herein and / or for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein. In alternative embodiments, the product of manufacture comprises or is fabricated as a biosensor or a microfluidic device. Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and / or Detailed Description sections. As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” PATENT 5810.152599PCT / Love-J7 encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court. Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims. The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples. PATENT 5810.152599PCT / Love-J7 EXAMPLES Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany. Example 1: Design of Protein Heterodimers Utilizing Metal-Ligand and Salt-Bridge Interactions This example describes how to make and use exemplary non-natural synthetic protein heterodimers as provided herein. In alternative embodiments, provided are methods that drive protein-protein interactions through engineered metal-ligand interactions and salt-bridge interactions using exemplary non-natural synthetic protein heterodimers as provided herein; this enables reversible association of two unique binding partners of the synthetic protein heterodimer. In alternative embodiments each component has designed interfaces which enhance association in the presence of exogenous agents such as divalent metal ions and dissociation with metal chelating agents. This design of chemically controlled asymmetric protein-protein interfacing will further enhance the building of precise protein-based biomaterials. Structure-based design is used to engineer three metal-controlled homodimer complexes that bind with high affinity in the presence of divalent zinc ions (Zn2+). To increase the versatility of these protein-based tools we converted one of the homodimers into a metal-controlled heterodimer. To achieve this, the homodimer was re-engineered to generate two distinct binding partners that only bind one another in the presence of metal ions. To promote specificity of the metal-controlled complex PATENT 5810.152599PCT / Love-J7 formation, and prevent self-association, it was necessary to incorporate elements of positive and negative design, which was achieved through the incorporation of a cross-interface electrostatic interaction as well as modifications to hydrophobic contacts at the protein-protein interface. The resulting metal-controlled heterodimer binds with low micromolar affinity, and the crystal structure indicates the presence of the designed dual-inter-action motifs at the protein-protein interface. Until recently, the only means of controlling protein-protein interactions with metal ions primarily entailed the use of metal-controlled homodimers6,12,11,4,9. Homodimers have limited utility unless the goal is to bring a fused passenger protein into close proximity with just itself. For example, if one fuses passenger protein A to a metal-controlled homodimer (MC), generating MCA, and passenger protein B to the same MC homodimer, generating MCB, then a heterogeneous mixture of dimers will result upon adding metal. Assuming the passenger protein has no effect on homodimer assembly, one would obtain a mixture of dimer structures that consist of 25% MCA / MCA, 25% MCB / MCB, 25% MCA / MCB, and 25% MCB / MCA, resulting in only 50% of the complexes in which passenger protein A is brought into close proximity with passenger protein B, see FIG.1. To address this issue, a metal-controlled heterodimer as provided herein is more useful because it results in dimer structures in which passenger protein A is only brought into proximity with protein B. This orthogonal interaction strategy using metal-controlled association allow for the programmable design of more precise protein-protein interactions. Therefore, the goal of this research was to reengineer a metal-controlled homodimer and convert it into a metal-controlled heterodimer. Three metal-controlled homodimer proteins were designed and biophysically characterized, which demonstrated that the proteins dimerize with high affinity in the presence of Zn2+ and dissociate upon the addition of EDTA12. For the work described herein, one of these metal-controlled homodimers was converted to a heterodimer, which dimerizes through both metal binding and a designed interfacial salt-bridge. Initial attempts at generating the heterodimer were not fully successful due to the fact that one of the binding partners exhibited propensity to self-associate. To eliminate the self-association, the degree of hydrophobic contact at the heterodimer interface was reduced via selective mutagenesis and resulted in the generation of a successful protein heterodimer. The engineered heterodimer was PATENT 5810.152599PCT / Love-J7 biophysically characterized, and its structure solved by x-ray crystallography, which revealed that the protein adopts the targeted heterodimeric structure, and does so, through metal binding and a designed cross-inter-face salt-bridge. Results and Discussion The homodimer designs were based, in part, on the crystal structures of Gβ1 mutant variants that are monomeric in the absence of metal yet form heterogeneous complexes in the presence of zinc12. A key intermolecular feature, revealed by the crystal structures of two such variants, is dimerization via extension of the Gβ1 β- sheet. This form of intermolecular interaction is the same observed for the crystal structure of a constitutive homodimer of Gβ1 (i.e., non-metal binding), which was generated via directed evolution by a different research group27. This variant, termed Gβ1-M2 (PDB entry 3FIL), is a fourfold mutant of Gβ1 (i.e., E15V, T16L, T18I, and N37L) that forms a symmetric homodimer through intermolecular extension of the β- sheet. Combinations of the four mutations, plus mutations to metal-binding histidine residues, were used to generate the three metal-controlled homodimers. Characterization of the homodimers revealed that they adopt the target dimer structure and bind with relative high affinity. The variant that binds with the highest affinity, MCD_C1 (MCD - metal-controlled dimer; PDB entry 6NL8), has a fluorescence- based analytical ultracentrifugation measured binding constant that is estimated to be less than 75 pM12. The protein interface of MCD_C1 consists of leucine residues at positions 16 and 37 and metal-coordinating histidine residues at positions 12, 29, and 33 (positions 15 and 18 were reverted back to wild type residues for the MCD_C1 variant). This variant was selected as the design scaffold for conversion to a heterodimer due to its high binding affinity. Figure 2. Illustration of Potential Hetero- and Homodimer Associations. A) Metal-binding histidine residues are illustrated for exemplary engineered symmetric homodimer (MCD_C1). B) Designed exemplary heterodimer (described herein) with one intact metal binding site at bottom and a designed intermolecular electrostatic interaction at top. Panels C & D represent unwanted homodimers that could form through self-binding of either of the heterodimer binding partners. C) R12H29H33 half (blue in B) or D) the H12E29E33 half (green in B) of the heterodimer variant. The metal-controlled homodimers bind symmetrically and thus contain two symmetry related metal coordination sites (Figure 2A). Each site is made up of three PATENT 5810.152599PCT / Love-J7 histidine residues: two at positions 29 and 33 (from one monomer) and a third at position 12 (from the other monomer). The inter-faces of symmetric protein complexes contain two sets of pair-wise interactions, and thus twice the number of energetically favorable interactions28. In addition, strong binding for protein dimers may be enhanced when hydrophobic residues are matched on interacting surfaces29. Relative to heterodimeric structures, homodimers are more energetically favorable as their symmetrical nature enhances self-association and interaction propensity29. Therefore, the conversion of a homodimer to a heterodimer was a more challenging process due to the loss of the symmetry-related contributions, and thus one expects concomitant loss of binding affinity upon reduction of favorable symmetry elements at the dimer interface. The engineering of a heterodimer protein pair necessitates the design of two different proteins that exhibit low to no self-binding while maintaining affinity for the binding partner. During evolution of natural heterodimers, negative and positive selection likely enabled the generation of stable heterodimers. An example of negative selection is the incorporation of like-charge residues across an unwanted homodimer interface that function to repel self-association. Conversely, the juxtaposition of oppositely charged amino acids across the heterodimer interface provides favorable binding enthalpy through electrostatic interactions. The presence of charged residues across the dimer interface represents both positive and negative elements for conversion to a heterodimer. To achieve this, it was necessary to break symmetry and convert one of the metal binding sites to an electrostatic interaction (Figure 2B). Salt bridges occur between two or more groups of oppositely charged residues within 4 Å30. A simple salt bridge is defined as an interaction between two oppositely charged residues, while a complex salt bridge involves three or more charged residues31. Many natural proteins utilize either a simple salt bridge or networks electrostatic interactions that function to stabilize protein structure or provide favor- able binding enthalpy at protein / protein interfaces. Complex salt bridges, referred to as ‘networked’, often involve electrostatic interactions between one basic residue and two acidic residues30,31. These types of interactions have been found to contribute to conformational stability, molecular recognition, or catalysis30. Our design goal was to create a heterodimer by engineering the following modes of interactions that bridge the heterodimer interface and prevent self- PATENT 5810.152599PCT / Love-J7 association: 1) each binding partner was designed to retain one metal binding site comprised of three histidine residues and 2) a ‘net-worked’ salt-bridge. Inspired by naturally networked salt bridges (i.e., a 2:1 ratio of acidic to basic sidechains) we con- verted one of the histidine residues to arginine and substituted the other two histidine residues for glutamic acid residues (Figure 2B, upper site). Arginine was used due to its prevalence in naturally occurring salt bridges, the greater number of possible hydrogen bonds, and delocalized charge density. In nature, arginine is found to be the most abundant charged residue at the interface of protein-protein interac-tions31,32. With many natural protein salt bridges, the prevalence of glutamic and aspartic acid are approximately equal, with a slight preference for glutamic acid31. We rationalized that the longer chain length of glutamic acid might promote more conformational freedom upon forming salt-bridge across the interface with arginine31. Figure 3. PYMOL Model of the Putative ‘Networked’ Electrostatic Salt Bridge. The X-ray structure of the MCD_C1 homodimer was used to model an arginine residue at position 12 and verify that it is within an electrostatic bond length with the glutamic acid residues at positions 29 and 33 of the corresponding binding partner. For the first monomer of the designed heterodimeric pair, His12 was mutated to arginine whereas positions 29 and 33 remained histidine residues (Figure 3, pink monomer on left). This variant, referred to as MCHet_A, also contains the two MCD_C1 derived interfacial leucine mutations at positions 16 and 37. For the associated binding partner, MCHet_B (Figure 3, (green) monomer on right), a histidine residue remained at position 12 whereas glutamic acid was introduced at positions 29 and 33. This variant also contains leucine residues at positions 16 and 37. These are being mentioned here because, in later steps, it was necessary to reduce the hydrophobic content of the dimer interface by altering the amino acid types at these residue positions. SEC MALS Characterization of Designed Heterodimeric Binding Proteins The MCHet_A and MCHet_B variants were expressed, purified, and analyzed using size exclusion chromatography (SEC) coupled to multi-angle light scattering (MALS) to assess formation of a heterodimer. Analysis of MCHet_A (R12H29H33), in the absence and presence of metal (1 mM zinc sulfate), revealed that it remains relatively monomeric in both states (Figure 4A). The SEC MALS (size exclusion PATENT 5810.152599PCT / Love-J7 chromatography with multi-angle light scattering) measured molecular weights for MCHet_A are 7.1 kD and 7.5 kD in the absence and presence of 1 mM zinc sulfate, respectively. Figure 4. SEC MALS Analysis of Single Heterodimer Variants and Complexes. Black curves correspond to samples with no added metal, while red curves correspond to samples that contain 1 mM Zn2+. A) MCHet_A by itself; B) MCHet_B by itself; and C) equimolar concentrations of MCHet_A and MCHet_B. In panel A the y-axis corresponds to molar absorbance, whereas in B and C the y-axis values were converted to molar mass in grams per mole. The x-axis corresponds to milliliters of solvent during the SEC run. As opposed to that observed for MCHet_A, analysis of MCHet_B (H12E29E33), analyzed alone, revealed that it is monomeric in the absence of metal (6.8 kD) yet forms a homodimer upon the addition of zinc (12.2 kD, Figure 4B). For MCHet_B, the two glutamic acid residues are in close proximity at helical positions 29 and 33. This bidentate pair could possibly contribute to homodimer formation through electrostatic interactions (potentially metal-mediated) be-tween His 12 on one monomer and the glutamic acid at positions 29 and 33 across the interface (Figure 2D). To determine if a heterodimer would preferentially form between MCHet_A and MCHet_B, a one-to-one molar ratio was analyzed in both the presence and absence of zinc sulfate. The theoretical molecular weight of a dimeric complex between these two variants is 12.4 kD. In the absence of metal, associations between MCHet_A and MCHet_B resulted in an average molecular weight of 11.1 kD, thus association occurs in the absence of metal (Figure 4C). For the sample in which metal was added, the complex exhibited a molecular weight of 13.4 kD. Thus, metal- independent interactions likely occur between MCHet_A and MCHet_B. The SEC MALS results indicated that metal-independent and possibly metal- mediated interactions occur between MCHet_A and MCHet_B, whereas when MCHet_A is analyzed by itself it is monomeric in the presence and absence of metal. If it did not bind MCHet_B, then a monomer peak for MCHet_A would have been observed. This is not the case and therefore it is likely that MCHet_B has a higher affinity for MCHet_A than for itself. Interestingly, MCHet_B appears to bind MCHet_A in the absence of metal, yet the parent homodimer, MCD_C1, does not PATENT 5810.152599PCT / Love-J7 homodimerize at all without metal. The only difference between MCD_C1 and MCHet_B are the substitutions of the two histidine residues at positions 29 and 33 for glutamic acid residues. For MCHet_A, relative to MCD_C1, there is a single mutation of arginine for histidine at position 12. To summarize, MCD_C1 does not homodimerize in the absence of metal, yet MCHet_A and MCHet_B do bind one another in the absence of metal. Therefore, it can be surmised that the designed electrostatic salt bridge between MCHet_A (H12R) and MCHet_B (H29, 33E) likely provides enough energy for the complex to form in the absence of metal, and also implies that the designed salt bridge provides favorable intermolecular binding en- ergy. Although this finding is interesting, the overall goal of generating non-self- associating monomers, which form a heterodimer in the presence of metal, was not achieved. To eliminate the self-association of MCHet_B, the degree of hydrophobicity at the dimer interface was reduced by mutating the two leucine residues at positions 16 and 37 to valines. Mutation of positions 16 and 37 to valines resulted in the creation of a third variant, referred to as MCHet_C, which is identical to MCHet_B except that it contains valine at positions 16 and 37 (i.e., MCHet_C = H12E29E33, V16&37). MCHet_C was expressed, purified, and subjected to SEC MALS analysis in the presence and absence of zinc. The results indicate that, although it is not perfectly monomeric in the presence of metal, its propensity to self-associate has been significantly reduced (Figure 5A). The SEC MALS measured molecular weights for MCHet_C are 6.9 kD and 8.3 kD in the absence and presence of zinc, respectively (Table 2). Figure 5. SEC MALS Analysis of Single Heterodimer Variants and Complexes. Red curves correspond to samples that contain 1 mM Zn2+, black curves correspond to samples with no added Zn2+. A) MCHet_C by itself; B) equimolar concentrations of MCHet_A and MCHet_C. In panel A the y-axis corresponds to molar absorbance, whereas in B the y-axis values were converted to molar mass in grams per mole. The x-axis corresponds to milliliters of solvent during the SEC run. Table 1: Mutations for the Parent Homodimer and Three Hetero-dimer Variants PATENT 5810.152599PCT / Love-J7 Table 2: SEC MALS Measured Molecular Weights of Individual and Complex Heterodimer Variants Table 3: Molecular Weight and Binding Affinity from Analytical Ultracentrifugation AUC Characterization of Exemplary Designed Heterodimeric Binding Proteins In addition to subjecting the metal-controlled heterodimers to SEC MALS analysis, the molecular weights in the presence and absence of zinc sulfate were also analyzed using two different analytical ultracentrifugation methods, i.e., PATENT 5810.152599PCT / Love-J7 sedimentation velocity (SV) and sedimentation equilibrium (SE). For the SV experiments molecules migrate as a function of size, shape, and interaction with other macromole-cules33 whereas in SE experiments the boundaries of each molecule separate until equilibrium is reached giving a distribution of associated and non- associated states. In sedimentation equilibrium one can accurately measure molecular weights, self-association, and hetero-association by fitting data obtained under different protein concentrations33. The MCHet_A / MCHet_C heterodimer complex was subjected to sedimentation velocity analysis to determine the molecular weights of each individual component in addition to interactions between the two proteins. When analyzed individually by SV the measured molecular weights indicate that each protein is monomeric in the absence and presence of zinc. When incubated together, and in the absence of zinc, no intermolecular interactions were observed (Supporting Information). Subsequent analysis of the two proteins in the presence of zinc sulfate indicated that a metal-controlled dimer forms between these two variants with an SV measured molecular weight of 11.7 kDa. For the SE experiments MCHet_A and MCHet_C were evaluated separately over a concentration range in the presence of zinc. MCHet_A remains monomeric and has an apparent molecular weight of 6.7 kDa. MCHet_C has an apparent molecular weight of 7.8 kDa, which is higher than the monomer molecular weight. Fitting the SE binding data for MCHet_C to a monomer-dimer equilibrium model yields fairly weak dissociation constants of 571 μM and 1,456 μM at rotor speeds of 30,000 and 35,000 rpm, respectively. When ana-lyzed together, and in the presence of zinc sulfate, the complexes formed by MCHet_A / MCHet_C has an apparent molecular weight of 11.7 kDa, and a binding affinity of approximately 18 and approximately 19 μM at the rotor speeds of 30,000 and 35,000 rpm, respectively. The affinity of the metal-controlled binding protein interaction was confirmed by similar dissociation constants at two different rotor speeds (data not shown). Salt Dependence Analysis on Heterodimer Formation The putative intermolecular salt bridge that potentially contributes binding energy to heterodimer formation con-sists of an arginine residue from MCHet_A and two cross-interface glutamic acid residues from MCHet_C. To confirm that the salt bridge contributes binding energy, dimer formation (in the presence of zinc) was PATENT 5810.152599PCT / Love-J7 disrupted by titration with increasing concentrations of sodium chloride. For this experiment all samples were subjected to SEC-MALS analysis using a buffer that consists of 20 mM Tris, 1 mM zinc sulfate, pH 7.0, and increasing concentrations of sodium chloride. The AUC determined dissociation constant for the metal-controlled heterodimer (MCHet_A / MCHet_C) is approximately 18-19 μM. Most all of the previous SEC-MALS analyses were performed using a protein concentration of 1 mM, which is approximately 50-fold greater than its dissociation constant. To ensure that the complexes were closer to a more ideal state of equilibrium, the concentrations for both binding partners were reduced to 100 μM, which is approximately 5 times greater than the AUC determined dis-sociation constant. Figure 6. SEC MALS measured Salt-Dependence on Heterodimer Formation. The five traces correspond to SEC MALS runs performed in increasing sodium chloride concentrations. Curves to the left indicate larger molecular entities and thus higher molecular weights. Heterodimer formation is disrupted upon increasing concentrations of sodium chloride as the retention time increases, indicating lower molecular weights. When the complexes were studied in a buffer containing 100 mM sodium chloride, and at the lower protein concentration (100 μM), the metal-controlled heterodimer has a SEC MALS measured molecular weight of 10.7 kDa, which is similar to the measured molecular weight of 12.0 kDa determined at a higher protein concentration (1 mM). As the salt concentration was increased, the measured molecular weight is reduced from a dimer value to that observed for the separate monomers (Figure 6). Thus, the MCHet_A / MCHet_C dimer complex is effectively rendered monomeric when incubated in either 1 M or 2 M sodium chloride. The results of this analysis suggest a salt dependence on complex formation and the designed salt-bridge likely provides favorable binding energy and contributes to greater intermolecular affinity. X-Ray Crystallographic Analysis of the MCHet_A / _B and MCHet_A / _C Heterodimer Complexes Crystals were grown for both the non-ideal heterodimer - MCHet_A / _B and the successful heterodimer - MCHet_A / _C. Both pairs readily form crystals in the presence of zinc. Upon collection and processing of high-resolution x-ray diffraction data, it was observed that the indexed collection of integrated reflection intensities for PATENT 5810.152599PCT / Love-J7 both crystals refine perfectly as if belonging to the body-centered tetragonal space group I41 with only one protomer (half of a heterodimer) per asymmetric unit. For example, systematic absences of reflections were observed as predicted in space group I41, even within the highest resolution spheres. We reasoned that both pairs of heterodimers (i.e., MCHet_A / _B and MCHet_A / _C) might have independently crystallized in the same I41 crystal system as a collection of heterodimers randomly oriented as either A:B or B:A. To test this conjecture, we reprocessed the data for the MCHet_A / _C heterodimer in triclinic P1 space group (unit cell dimensions a = 25.69 Å, b = 50.82 Å, c = 50.82 Å and α =86.3°, β = 75.4°, and γ = 75.4°) with two dimers per asymmetric unit. Molecular replacement and initial model refinement yielded electron density maps that contained clear convolutions of both side chains at sites where differences exist between the two protomers. This was also the case for other low symmetry space groups tested. Table 4: Molecular Weight Dependence on Salt Concentration Determined by SEC- MALS In order to accurately reflect the random packing orientation of the heterodimers, the final MCHet_A / _B and MCHet_A / _C models were built and refined with anisotropic B factors and riding hydrogens in space group I41 as two superposed protomers, each with occupancy of 0.5, and containing the amino acid sequences of either MCHet_A / _B (PDB ID: XXXX), or MCHet_A / _C (PDB ID: XXXX). The resulting models are of excellent quality (model and refinement statistics in Supporting Information - Table 4) and models for the heterodimers can be PATENT 5810.152599PCT / Love-J7 generated by rotating one protomer 180° about the z-axis relative to the other. Due to the extremely low level of chemical differences between protomers and consistent with random orientation of close packed heterodimers in the crystal, we observe practically identical patterns of atomic disorder, as judged by anisotropic B factors versus amino acid position, between protomer subunits of each heterodimer. The resulting structures not surprisingly reveal that the heterodimers take on the overall structure of the parent variant, MCD_C1 (Figure 7). Both complexes adopt a head-to-tail intermolecular orientation in which each heterodimer is formed through extension of the β-sheets. The structures and cross-interface hydrogen bond register for the constitutive homodimer - Gβ1-M2, the highest affinity metal-controlled homodimer - MCD_C1, and the heterodimer complexes - MCHet_A / _B and MCHet_A / _C, are all highly similar. For each structure, there are six interfacial hydrogen bonds: two between positions 17 and 13′, two between positions 15 and 15′, and two between positions 13 and 17′ (the numbering for cross-interface positions is de-noted with a prime). Figure 7. Crystal Structures of the Designed Heterodimer Com-plexes. (A) MCHet_A / _B and (B) MCHet_A / _C. The designed metal-ligand histidine residues and the residues involved in the salt bridge are illustrated as stick bonds, with the corresponding 2Fo – Fc omit electron density map (1.5σ). Zinc atoms are illustrated as amber spheres, and chlorine atoms as light grey spheres. The designed zinc coordination sites for both heterodimers are formed by two histidine residues in i to i + 4 positions on MCHet_A (His29 and His33) located on the central α-helix and an additional cross-interface interaction involving a designed histidine at position 12 (located on MCHet_B and MCHet_C), which is located on the turn between the first and second β-strands. Analogous to MCD_C1, the zinc coordination involves three histidine ligands and a chloride ion. Analysis of the heterodimer structures demonstrate that the distances and bond angles between the zinc atom and the three histidine ligands are within range of standard tetrahedral coordination geometry. In addition to the interface bridging zinc binding site, the interfaces between MCHet_A / _B and MCHet_A / _C were designed with networked salt bridges that consists of an argi-nine residue from one monomer and two glutamic acid residues from the other. The crystal structures of both complexes revealed an unanticipated PATENT 5810.152599PCT / Love-J7 finding - a second zinc atom (and associated chlorine counter ion) located in close proximity to the designed electrostatic salt bridge which appear to possibly contribute to this intermolecular interaction. For both complexes it appears that Glu 33 is positioned to interact electrostatically more directly with the zinc atom whereas Glu 29 is positioned to form both a favorable electrostatic interaction with the zinc atom and is also in proximity to bind electrostatically with the cross-interface Arg 12 residue. The arginine guanidinium group provides five possible sites for hydrogen bonding interactions and analysis of the MCHet_A / MCHet_B crystal structure reveals that Glu 29 is positioned to potentially form two hydrogen bonds with the terminal Nη and Nε atoms of the arginine side chain. In addition, the arginine side chain is potentially stabilized through a possible hydrogen bond with Glu 33. The protein-protein interface of the MCHet_A / _C dimer contains a designed intermolecular salt bridge at one site and zinc coordination at the other. In a manner similar to the MCHet_A / _B heterodimer, the crystal structure MCHet_A / _C also indicates the presence of a zinc atom that appears to possibly function to mediate the salt bridge. There are three potential intermolecular hydrogen bonding bonds between Arg 12 and Glu 29 and 33. A similar hydrogen bonding pattern is observed in both heterodimer structures, but the hydrogen bond distances are slightly longer for the MCHet_A / _C heterodimer. Variations in the hydrogen bonding distances could possibly be due the weaker hydrophobic contact at the interface as well as to different crystallization conditions and / or variable crystal packing forces. As described above, MCHet_B was redesigned to reduce its propensity to self- associate, which lead to MCHet_C. This was accomplished by reducing the hydrophobicity at the interface by replacing leucine residues at positions 16 and 37 with valine residues. The resulting monomer, MCHet_C, exhibits reduced affinity for itself and thus remains relatively monomeric in the presence or absence of metal ions. Analysis of the MCHet_A / _B and MCHet_A / _C crystal structures reveals that the design goal of limiting hydrophobic contact at the interface was achieved. This is graphically illustrated in Figure 8 where a greater degree of hydrophobic contact is observed at the interface of MCHet_A / _B (Figure 8A) relative to that shown for the MCHet_A / _C complex (Figure 8B). Figure 8. Comparison of the Interfacial Hydrophobic Contacts. For the MCHet_A / _B complex (A) all four interfacial leucine residues are colored yellow. PATENT 5810.152599PCT / Love-J7 For the MCHet_A / _C complex (B) the two leucine residues from MCHet_A are colored yellow (left) whereas the valine residues from MCHet_C (right) are colored green. Semitransparent gray spheres representing van der Waals radii for the leucine residues (A) and for the valine residues (B) are illustrated for MCHet_B and MCHet_C respectively. In summary, we converted an engineered metal-ligand inducible homodimer into a heterodimer. This was achieved by breaking symmetry and converting one of the homodimer metal binding sites into an intermolecular salt-bridge. To reduce the propensity of one of the resulting heterodimer partners to self-associate the degree of hydro-phobic interaction was reduced at the designed interface. This resulted in a heterodimer pair of binding partners that have little to no propensity to self-associate and preferentially bind one another. The crystal structures of the resulting complexes indicate that the overall binding mode is maintained (i.e., antiparallel intermolecular extension of the β-sheets) and that the designed electrostatic interaction likely contributes favorable enthalpy to complex formation. The successful MCHet_A / _C complex can now potentially be used as a biotechnological tool to bring fused passenger proteins together and thus further enhance the ability to build novel molecular complexes. Methods Gene Synthesis, Protein Expression, and Purification The genes for the MCHet_A, MCHet_B, and MCHet_C variants were synthesized using four overlapping oligonucleotides and standard PCR methods. The genes were cloned in the pet21a expression vector (Novagen) using restriction sites EcoR1 and Nde1. E. coli strain BL21(DE3) was chemically transformed with the corresponding plasmids and grown to an OD600 approximately 0.8. Protein expression was induced for ~3 hours upon addition of IPTG at a final concentration of 1 mM. Cells were pelleted by centrifugation at 6,000 rpm for 10 minutes and stored in a -80 °C freezer. A freeze-thaw protein extraction cycle was utilized and included incubation on ice for 30 minutes, room temperature for 15 minutes, followed with a dry-ice ethanol bath for 10 minutes, and repeated three times. The cell pellet was gently resuspended in PBS, pH 6.8 for 60 minutes. The sample was centrifuged at 10,000 rpm for 30 minutes and acetonitrile was added to the resulting supernatant (to 30% v / v) to induce precipitation of impurities. The resulting supernatant was PATENT 5810.152599PCT / Love-J7 lyophilized to remove the acetonitrile-water. The lyophilized sample was resuspended in water and purified using a Varian 10-micron C8 HPLC preparative reverse-phased column with a linear 1% / min acetonitrile / water gradient containing 0.1% trifluoroacetic acid. The purified protein was lyophilized, and the resulting dry protein sus-pended in water. The pH was adjusted to approximately 7 and buffered with 20 mM Tris, 100 mM sodium chloride, 200 ppm sodium azide, and pH 7.0. Protein purify was assessed using SDS-PAGE and protein concentration determined by absorbance at 280 nm using calculated calculated / theoretical extinction coefficients. The proteins were concentrated using centrifugal filtration to approximately 2 mM (approximately 12 mg / mL) and stored at -20oC. SEC MALS Characterization Stock proteins of the heterodimer variants, and controls were diluted to 1 mM in buffer containing the following four sets of solution components: 1) 20 mM Tris, 100 mM sodium chloride, pH 7.0; 2) 20 mM Tris, 100 mM sodium chloride, 5 mM EDTA, pH 7.0; 3) 20 mM Tris, 100 mM sodium chloride, 500 μM zinc sulfate, pH 7.0; 4) 20 mM Tris, 100 mM sodium chloride, 1 mM zinc sulfate, pH 7.0. Size exclusion chromatography was performed using a FPLC (GE Healthcare) with UV detection at 280 nm. The FPLC was connected in-line with multiangle light scattering (Wyatt miniDAWN TREOS detector system). ASTRA software (Wyatt Technologies) was used to analyze elution peak(s) and determine the molecular weight calculated by light scattering. X-ray Crystallography For crystallization purposes, MCHet_A, MCHet_B, and MCHet_C were expressed and purified as described above and concentrated to a minimum 2 mM. To study the effects of metal-controlled association, excess zinc sulfate was added to an equimolar solution containing 1 mM of MCHet_A and MCHet_B and MCHet_A and MCHet_C prior to crystallization. Crystals were obtained by vapor diffusion at room temperature from hanging drops. The 3 μL drop consisted of 2 mM Protein (1 mM MCHet_A and 1 mM MCHet_B or MCHet_C incubated with 4 mM zinc sulfate) was mixed with 3 μL of the crystallization solution. The optimized crystallography conditions for binding MCHet_A / MCHet_B and MCHet_A / MCHet_C were 0.1 M HEPES pH 7.5200 mM MgCl224 % PEG 3000 and 0.1 M HEPES pH 7.5200 mM MgCl226 % PEG 10000, respectively. Optimized crystallization buffer with 5% PATENT 5810.152599PCT / Love-J7 glycerol was used for storage and transport in liquid nitrogen. Data were collected at the Berkley Lab Advanced Light Source (ALS-5.0.1™). Data were processed using the programs XDS34 and iMOSFLM34™. Structures were determined by molecular replacement with the program PHENIX34™. The protein Gβ1 domain (PDB entry 1PGA) was used as a molecular replacement model. The refinement was carried out with PHENIX refine using several cycles of the least-squares refinement fit interspaced with manual editing of the structure in COOT35. The refinement procedure usually converged with fewer than 10 macrocycles of the refinement and manual inspection. The structures were analyzed using PISA36 and MolProbity37. The structures are currently being prepared for submission to the Protein Data Bank (PDB). Some additional analysis of the metal ion-binding sites was performed using the CHECKMYMETAL (CMM)38™ online server. Descriptive software tools such as PISA and CCM provide an estimate for binding energies and a secondary validation tool for organization of the lattice and the binding sites. Data collection and refinement statistics are listed in (Table 4). Analytical Ultracentrifugation: Sedimentation Velocity MCHet_A and MCHet_C were subjected to sedimentation velocity analytical ultracentrifugation analysis at the Sanford Burnham Prebys Medical Discovery Institute. MCHet_A and MCHet_C were individually prepared in 20 mM Tris, 100 mM sodium chloride, pH 7.0- and 20-mM Tris, 100 mM sodium chloride, 25 μM zinc sulfate, pH 7.0. Samples were analyzed using a Beckman Coulter ProteomeLab XL- 1™ analytical ultracentrifuge equipped with UV-absorption detector. Samples were loaded into both sectors of double sector cell equipped with sapphire windows. Data were collected at 21 ºC for 24 hours at a rotor speed of 42,000 rpm with a 50 Ti rotor. Data were analyzed in Sedfit39 using the c(s) model. The molecular masses, partial specific volumes, solvent density and viscosity used in the data analysis were calculated in SEDNTERP™40. Analytical Ultracentrifugation: Sedimentation Equilibrium Sedimentation equilibrium experiments were performed using a Beckman Coulter ProteomeLab XL-I™ analytical ultra-centrifuge at the Sanford Burnham Prebys Medical Discovery Institute. Samples were prepared in 20 mM Tris, 100 mM sodium chloride, 60 μM zinc sulfate, pH 7.0 buffer. Protein samples at concentrations 40 μM, 20 μM, and 10 μM were loaded in a 6-channel equilibrium cells and spun in PATENT 5810.152599PCT / Love-J7 an An-50 Ti Analytical 8-place titanium rotor at 21oC until equilibrium was reached. Data sets for MCHet_A, MCHet_C analyzed alone and then with equimolar concentration so both proteins were collected at 30,000 rpm and 35,000 rpm. HeteroAnalysis AUC™ software was used for the data analysis of the binding partners. The analytical ultracentrifugation data sets determining apparent molecular weight were fitted to the single ideal species model. The data sets deter-mining the association constant were fitted to a monomer-dimer model. Amino Acid Sequences of Gβ1 and its variants Amino Acid Sequences for SEQ Sequence Metal-Controlled Dimer and ID Binding Partners Mutations NO: Gβ1 wild-type SEQ MTYKLILNGKTLKGETTTEAVDA ID ATAEKVFKQYANDNGVDGEWT NO:1 YDDATKTFTVTE Metal- L12H, T16L, SEQ MTYKLILNGKTHKGELTTEAVD Controlled V29H, Y33H, ID AATAEKHFKQHANDLGVDGEW Homodimer N37L NO:2 TYDDATKTFTVTE Metal- SEQ Controlled ID AATAEKHFKQHANDAGVDGE Homodimer NO:6 WTYDDATKTFTVTE Metal- SEQ MTYKLILNGKTHKGVLTIEAVD Controlled ID AATAEKHFKQHANDLGVDGE Homodimer NO:7 WTYDDATKTFTVTE MCHet_A L12R, T16L, SEQ MTYKLILNGKTRKGELTTEAVDA V29H, Y33H, ID ATAEKHFKQHANDLGVDGEWT N37L NO:3 YDDATKTFTVTE MCHet_B L12H, T16L, SEQ MTYKLILNGKTHKGELTTEAVD V29E, Y33E, ID AATAEKEFKQEANDLGVDGEWT N37V NO:4 YDDATKTFTVTE MCHet_C L12H, T16V, SEQ MTYKLILNGKTHKGEVTTEAVD V29E, Y33E, ID AATAEKEFKQEANDVGVDGEWT N37V NO:5 YDDATKTFTVTE In alternative embodiments, provided are transition Metal Binding Sequences for the Three Metal Controlled Dimers (MCD), including proteins having the amino acid sequences SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7, which have been shown to bind the transition metals zinc, nickel, and cobalt. In alternative embodiments, provided are sequences that correspond to the generation of heterodimer pairs of proteins, which are different from the MCDs which form homodimers: all three of these proteins are monomeric in the absence of metals such as zinc, where MCHet_A (SEQ ID NO:3) forms a heterodimer with MCHet_B PATENT 5810.152599PCT / Love-J7 (SEQ ID NO:4) and also MCHet_C (SEQ ID NO:5) in the presence of zin; but MCHet_B (SEQ ID NO:4) was demonstrated to self-associate to some degree in the presence of zinc. MCHet_C (SEQ ID NO:5) was demonstrated to not self-associate in the presence of zinc. In summary, MCHet_A (SEQ ID NO:3) forms a clean heterodimer with MCHet_C (SEQ ID NO:4); therefore, these proteins have potential application as critical components in different biotechnological tools. In alternative embodiments, provided are Rare Earth Binding Proteins (REE_BPs); below is a list of different exemplary protein-G variants that have been designed to bind Rare Earth Elements (REEs) (referred to as Rare Earth Element Binding Proteins (REE-BPs): REE_BP_2D3E MTYKLILNGKEDKGELTEEAVDAATAEKDFKQEANDLGVDGEWTYDDATK TFTVTE (SEQ ID NO:8) REE_BP_All_Glu MTYKLILNGKEEKGELTEEAVDAATAEKEFKQEANDLGVDGEWTYDDATKT FTVTE (SEQ ID NO:9) REE_BP_All_Glu_Loop MTYKLILNGKAGPEDKGELTEEAVDAATAEKEFKQEANDLGVDGEWTYDD ATKTFTVTE (SEQ ID NO:10) REE_BP_All_Glu_Loop_extra_G MTYKLILNGKAGPEGDKGELTEEAVDAATAEKEFKQEANDLGVDGEWTYD DATKTFTVTE (SEQ ID NO:11) In addition to generating these individual proteins and characterizing them using size exclusion chromatography (SEC) combined with multi-in the light scattering (MALS), we have also generated the following mutants that are the same sequences as the parent sequences above, but now they also have the following additional sequence of amino acids added to the C terminus (GGSGGCC) (SEQ ID NO:41). The purpose of adding this amino acid sequence to the C terminus is so that, after expression and purification, the proteins can be bio-conjugated to gold nanoparticles for the purpose of additional biotechnological uses and detection methods. Bioconjugation to gold nanoparticles has already been successfully demonstrated and characterized using different forms of spectroscopy. MCD_C1_AuNP MTYKLILNGKTHKGELTTEAVDAATAEKHFKQHANDLGVDGEWTYDDATK TFTVTEGGSGGCC (SEQ ID NO:12) PATENT 5810.152599PCT / Love-J7 MCD_M2_AuNP MTYKLILNGKTHKGVLTIEAVDAATAEKHFKQHANDLGVDGEWTYDDATK TFTVTEGGSGGCC (SEQ ID NO:13) REE_BP_1D4E_loop_AuNP MTYKLILNGKAGPEDKGELTEEAVDAATAEKEFKQEANDLGVDGEWTYDDA TKTFTVTEGGSGGCC (SEQ ID NO:14) The following series of four mutant variants were designed and produced with the goal of mutating all non-rare earth element binding ligand sidechains from glutamic acid and aspartic acid to glutamine (Q) and asparagine (N), respectively. These variants are referred to as the QN variants: QN variants REE_2D3E_QN MTYKLILNGKEDKGQLTEQAVNAATAQKDFKQEANNLGVNGQWTYNNATK TFTVTQ (SEQ ID NO:15) REE_AllGlu_QN MTYKLILNGKEEKGQLTEQAVNAATAQKEFKQEANNLGVNGQWTYNNATK TFTVTQ (SEQ ID NO:16) REE_Loop (1D_4E) MTYKLILNGKAGPEDKGQLTEQAVNAATAQKEFKQEANNLGVNGQWTYNN ATKTFTVTQ (SEQ ID NO:17) REE_ExG (1D_4E) MTYKLILNGKAGPEGDKGQLTEQAVNAATAQKEFKQEANNLGVNGQWTYN NATKTFTVTQ (SEQ ID NO:18) The mutations made to the QN variants described above completely reduced the ability to express these proteins in the E. coli expression system used successfully many times in the past to express these proteins. Therefore, the following series of mutations were designed and tested in which 3 / 10, 4 / 10, 5 / 10, and 6 / 10 of the mutated glutamine and asparagine residues sidechains were returned to the wild type amino acids, glutamic acid and aspartic acid. These variants were all tested on the All_Glu variant described above. These four variants are generally referred to as the ‘hedgehog repair’ variants. Hedgehog repair All_Glu 3 / 10 MTYKLILNGKEEKGELTEQAVNAATAQKEFKQEANNLGVNGQWTYDNAT KTFTVTE (SEQ ID NO:19) 4 / 10 PATENT 5810.152599PCT / Love-J7 MTYKLILNGKEEKGELTEQAVNAATAQKEFKQEANDLGVNGQWTYDNAT KTFTVTE (SEQ ID NO:20) 5 / 10 MTYKLILNGKEEKGELTEQAVNAATAEKEFKQEANDLGVNGQWTYDNAT KTFTVTE (SEQ ID NO:21) 6 / 10 MTYKLILNGKEEKGELTEQAVNAATAEKEFKQEANDLGVNGEWTYDNATK TFTVTE (SEQ ID NO:22) To improve the binding constants and binding specificity for Rare Earth Elements (REEs), a series of additional peptides were designed to improve the two described binding features: New_E / D_37 variants V15V36_2D3E MTYKLILNGKTDKGVLTEQAVNAATAQKEFKQEANVDGVDGEWTYDDATK TFTVTE (SEQ ID NO:23) V15I36_3D2E MTYKLILNGKTDKGVLTEQAVNAATAQKEFKQEANIDGVDGEWTYDDATK TFTVTE (SEQ ID NO:24) V15V36_AllGlu MTYKLILNGKTEKGVLTEQAVNAATAQKEFKQEANVEGVDGEWTYDDATK TFTVTE (SEQ ID NO:25) V15I36_AllGlu MTYKLILNGKTEKGVLTEQAVNAATAQKEFKQEANIEGVDGEWTYDDATKT FTVTE (SEQ ID NO:26) F36 variants V15F36_D37 Twist pET21 MTYKLILNGKTEKGVLTEQAVNAATAQKEFKQEANFDGVDGEWTYDDATK TFTVTE SEQ ID NO:27 V15F36_D33 Twist pET21 MTYKLILNGKTEKGVLTEQAVNAATAQKEFKQDANFEGVDGEWTYDDATK TFTVTE SEQ ID NO:28 Further exemplary peptides as provided herein: G14E variants nREE_E14_2D3E MTYKLILNGKTEKEQLTDQAVNAATAQKDFKQEANNLGVDGEWTYDNATK TFTVTE SEQ ID NO:29 PATENT 5810.152599PCT / Love-J7 nREE_E14_AllGlu MTYKLILNGKTEKEQLTEQAVNAATAQKEFKQEANNLGVDGEWTYDNATK TFTVTE SEQ ID NO:30 nREE_E14G15in_AllGlu MTYKLILNGKTEKEGQLTEQAVNAATAQKEFKQEANNLGVDGEWTYDNAT KTFTVTE SEQ ID NO:31 nREE_E14V15_AllGlu MTYKLILNGKTEKEVLTEQAVNAATAQKEFKQEANNLGVDGEWTYDNATK TFTVTE SEQ ID NO:32 G38E variants MTYKLILNGKTEKGQLTEQAVNAATAQKEFKQEANDLEVDGEWTYDNATK TFTVTE SEQ ID NO:34 MTYKLILNGKTEKGQLTEQAVNAATAQKEFKQEANDLEGVDGEWTYDNAT KTFTVTE SEQ ID NO:35 MTYKLILNGKTEKGQLTDQAVNAATAQKDFKQEANDLEGVDGEWTYDNAT KTFTVTE (SEQ ID NO:36) MTYKLILNGKTEKGQLTDQAVNAATAQKDFKQEANDLEVDGEWTYDNATK TFTVTE (SEQ ID NO:37) MTYKLILNGKTEKGVLTEQAVNAATAQKEFKQEANDLEVDGEWTYDNATK TFTVTE (SEQ ID NO:38) MTYKLILNGKTEKGVLTDQAVNAATAQKDFKQEANDLEVDGEWTYDNATK TFTVTE (SEQ ID NO:39) MTYKLILNGKTEKEQLTDQAVNAATAQKDFKQEANDLEVDGEWTYDNATK TFTVTE (SEQ ID NO:40) Directed Evolution for increased REE binding affinity and greater REE specificity In alternative embodiments, provided is a Directed Evolution process to generate RRE_BPs that bind REEs with higher affinity and greater specificity. In alternative embodiments, Directed Evolution processes as provided herein use a biotechnological tool comprising a Bacterial Service Display (BSD) system. In alternative embodiments, the BSD system comprises use of in-frame fusing of four proteins that constitute the BSD system. The first protein is the PelB leader PATENT 5810.152599PCT / Love-J7 sequence that directs the entire protein construct to the bacterial periplasmic space. Following expression and direction to the periplasmic space, the 22 amino acid PelB leader sequence is excised (cleaved off) by an endogenous bacterial enzyme and then the entire construct is displayed externally on the outer E. coli membrane. This leaves the Protein of Interest (POI) as the protein that is furthest extended out on the E. coli outer membrane. FIG.12 illustrates an exemplary Bacterial Service Display (BSD), where the variant, REE_BP_1D_4E_loop, is the POI and is shown to be displayed as the terminal protein in the three fused proteins inherent to the system (the 22 amino acid PelB leader sequence does not get displayed as it is excised). The fluorescent protein mCherry is included as the central protein of the construct for optimization of surface expression. A truncated version of the EstA transmembrane protein, which originated from the bacterial genome of Pseudomonas aeruginosa, is used to anchor the entire construct in the E. coli outer membrane. For the purposes of Directed Evolution, not one but two REE-BPs are displayed in the context of the multiple domain protein construct as illustrated in FIG. 12A. This construct contains a split fluorescent protein that will not fluoresce unless the two REE-BPs bind an REE, which will induce the REE-BPs to dimerize and then also bring the two split parts (strands) of the fluorescent protein into close proximity where they will recombine and fluoresce (image on right, next page). The entire construct illustrated FIG.12A will be substituted in as the Protein of Interest (POI) in the context of the Bacterial Surface Display (BSD) system. A library of different REE-BPs of appropriate diverse complexity will be generated and displayed on the surface of E. coli. To create appropriate diversity in the displayed protein library, select interfacial amino acid positions of the REE-BPs will be subjected to a semi- biased randomization method (described below) during the gene synthesis of the REE-BPs. In alternative embodiment, two copies of an REE_BP are expressed as the pink and green protein elements FIG.12A. During gene synthesis of the two REE- BPs, select codons for interfacial amino acid positions will be subjected to semi- biased randomization. For this purpose, REE-BP positions 11, 12, 14, 18, 19, 29, and 33 will be subjected to a semi-biased randomization process during DNA gene synthesis. The semi-biased randomization process is meant to only substitute the following amino acids at the seven positions listed above: His, Gln, Arg, Asn, Lys, PATENT 5810.152599PCT / Love-J7 Ser, Asp, Glu, and Gly. To achieve this, the first base position of the codons for these positions will be randomized with the DNA bases C, A, or G (and not T); the second position will be randomized with either A or G (not C or T); and the third codon position will be randomized with all four DNA bases (A, C, G, T). In addition, two other positions (15 and 36) will also be subjected to semi-biased randomization using any of the four DNA bases in the first codon position: only C and T in the second position (not A or G): and any of the four DNA bases in the third position. This will function to randomly include the following relatively hydrophobic amino acids at those two positions (15 and 36): Phe, Leu, Ser, Pro, Ile, Met, Thr, Val, and Ala. Once the genes for these test variants, which will contain codons that have been subjected to the semi-biased randomization method, are synthesized / purchased from a gene synthesis company, they will be cloned into the BSD system in order to express these two variants on the outer membrane of E. coli. Prior to adding any REE metals, the bacteria will be allowed to grow to a predetermined density and the resulting library will be subjected to fluorescence activated cell sorting (FACS) in order to select for any variants that might form constitutive dimers (proteins that randomly happen to have the amino acids that might drive them to dimerize in the absence of metals). After all of the self-associating variants are removed from the library using a first round of FACS, the library will then be split into separate flasks and individually incubated with single rare earth elements such as neodymium, lanthanum, dysprosium, gadolinium, etc. Again, the variants that randomly happen to have the correct amino acids for REE binding will bind the rare earth elements, induce fluorescence, and be selected for using FACS technology. SDS-PAGE analysis of Binding Partners The purity of the Binding Partners was assessed using SDS-PAGE. An 18% SDS_PAGE gel was used to analyze the individual components. The migration of the individual proteins do not correspond to the theoretical molecular weight of around 6.5 kDa. See FIG.9, illustrating the 18% SDS-PAGE Gel of MCHet_A, MCHet_B, and MCHet_C. Lane 1 – molecular weight markers; Lane 2 – MCHet_B; Lane 3 – MCHet_A; Lane 4 – MCHet_C. PATENT 5810.152599PCT / Love-J7 Thermal Denaturation of all Variants Monitored with Circular Dichroism The thermal denaturation melting temperatures for the heterodimer binding partners were measured using circular dichroism. The increase in the CD signal at 222 nm was monitored as a function of increasing temperature under different experimental conditions. Normalized curves with both the folded and unfolded states for the MCHet variants are shown in FIG.10. \Analytical Ultracentrifugation: Sedimentation Velocity of MCHet_A and MCHet_C The molecular weights in the presence and absence of zinc sulfate were also analyzed using two different analytical ultracentrifugation methods, i.e., sedimentation velocity (SV) and sedimentation equilibrium (SE); see FIG.11. Physical Characteristics of the Zinc Tetrahedral Coordination Site Zinc binding sites found in protein structures show that standard zinc nitrogen (histidine) distances in zinc metalloproteins is 2.09 ± 0.14 Å41. The crystal structures for MCHet_A and MCHet_B and MCHet_A and MCHet_C each contains four molecules in the asymmetric unit. The biological assembly of MCHet_A / MCHet_B and MCHet_A / MCHet_C are dimers, in the presence of zinc (II). Distances between each Zinc Atom and the Side-Chain Nitrogen Atom Angles at Zinc Atoms between pairs of Metal-Ligand Histidine Residues

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B.; et al. MolProbity: All-Atom Structure Validation for Macromolecular Crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr.2010, 66 (1), 12–21. (38) Zheng, H.; et al. CheckMyMetal: A Macromolecular Metal-Binding Validation Tool. Acta Crystallogr. Sect. D Struct. Biol.2017, 73, 223–233. (39) Schuck, P. Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophys. J.2000, 78 (3), 1606–1619. (40) Laue, T.; et al. Analytical Ultracentrifugation in Biochemistry and Polymer Science. R. Soc. Chem. Ed Harding S, Rowe A Hort. J.1992. PATENT 5810.152599PCT / Love-J7 A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

PATENT 5810.152599PCT / Love-J7 WHAT IS CLAIMED IS:

1. A synthetic, non-natural protein heterodimer comprising a first domain protein or peptide and a second domain protein or peptide, wherein the first domain protein or peptide and the second protein or peptide are each independently fused, coupled or joined to a half, a section of or a portion of a detectable moiety or entity, and wherein both the first domain protein or peptide and the second protein or peptide are independently capable of specifically binding to or associating with a (the same) target compound or composition, and the target compound or composition comprises: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB),PATENT 5810.152599PCT / Love-J7 mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, and when the first domain protein or peptide and the second domain protein or peptide each bind or are associated with to the same target compound or composition under aqueous conditions the half, section of or portion of the detectable moiety or entity on the first domain protein or peptide is brought into sufficient proximity to the half, section of or portion of the detectable moiety or entity on the second domain protein or peptide to initiate generation of, or generates, a detectable signal, or to generate a molecule that can generate a detectable signal, and when the first domain protein or peptide and the second domain protein or peptide are in aqueous solution without but not binding to or associated with the target compound or composition no detectable signal is generated, wherein optionally the first domain protein or peptide and the second domain protein or peptide each bind or are associated with to the same target compound or composition by electrostatic attraction or interaction, or by an electrostatic salt bridge, and optionally the first domain protein or peptide and the second domain protein or peptide comprise amino acids that are negatively charged in an aqueous solution and the target compound or composition is or comprises a positively charged ion, optionally a positively charged metal ion.

2. The synthetic, non-natural protein heterodimer of claim 1, wherein the first domain protein or peptide and the second domain protein or peptide comprise aPATENT 5810.152599PCT / Love-J7 secondary structural configuration comprising an antiparallel intermolecular extension of β-sheet.

3. The synthetic, non-natural protein heterodimer of claim 1, wherein the first domain protein comprises a sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

4. The synthetic, non-natural protein heterodimer of claim 3, wherein the heterodimer comprises: the pair SEQ ID NO:3 and SEQ ID NO:4; or, SEQ ID NO:3 and SEQ ID NO:

5.

5. The synthetic, non-natural protein heterodimer of any of claims 1 to claim 4, wherein the first domain protein or peptide and the second protein or peptide are each independently covalently linked or joined to the half, section of or portion of the detectable moiety or entity.

6. The synthetic, non-natural protein heterodimer of any of claims 1 to claim 5, wherein the detectable signal comprises a fluorescent signal.

7. A synthetic or non-natural protein complex comprising: a synthetic, non-natural protein heterodimer of any of claims 1 to 6, and: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercury (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid,PATENT 5810.152599PCT / Love-J7 an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin, wherein both the first domain protein or peptide and the second protein or peptide are independently specifically binding (or bound to) the target compound or composition.

8. The synthetic or non-natural protein complex of claim 7, wherein the two second domain proteins gain a function when the two first domain proteins specifically bind to each other, and optionally the function comprises an enzymatic activity, a binding ability or results in a signaling or a fluorescent effect.

9. A cell comprising a synthetic or non-natural protein complex of claim 7 or claim 8, or a synthetic, non-natural protein heterodimer of any of claims 1 to 6,PATENT 5810.152599PCT / Love-J7 wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell.

10. A product of manufacture, or a kit, comprising or having contained therein a synthetic or non-natural protein complex of claim 7, or a synthetic, non- natural protein heterodimer of any of claims 1 to claim 6, or a cell of claim 9, and optionally the product of manufacture is fabricated as a microfluidic device or a biosensor.

11. A method for detecting a target compound or a composition in an aqueous solution, comprising: (a) providing a synthetic, non-natural protein heterodimer of any of claims 1 to claim 6, wherein both the first domain protein or peptide and the second protein or peptide of the synthetic, non-natural protein heterodimer are independently capable of specifically binding to the same target compound or composition; (b) adding the synthetic, non-natural protein heterodimer of (a) to an aqueous solution; and (c) detecting and / or measuring whether, or not, a detectable signal is generated, wherein detection of a detectable signal indicates that the target compound or composition is present in the aqueous solution, and optionally the aqueous solution is a physiologic solution or saline.

12. The method of claim 11, further comprising adding to the solution a metal chelating compound, wherein optionally the metal chelating compound comprises EDTA, resulting in the chelation of a metal or metal ion bound to the synthetic, non-natural protein heterodimer, resulting in dissociation of the specific binding of the first domain protein or peptide and the second protein or peptide of the synthetic, non-natural protein heterodimer to the target compound or composition, and loss of generation of the detectable signal.

13. A recombinant or synthetic nucleic acid encoding SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.PATENT 5810.152599PCT / Love-J7 14. An expression vehicle comprising a recombinant or synthetic nucleic acid of claim 13, wherein optionally the expression vehicle is a viral particle, a recombinant virus, a baculovirus, a phage, a plasmid, a phagemid, a cosmid, a fosmid, a bacterial artificial chromosome, a viral DNA, or a P1-based artificial chromosomes.

15. A cell having contained therein, or comprising, a recombinant or synthetic nucleic acid of claim 13, or an expression vehicle of claim 14, or a synthetic, non-natural protein heterodimer of any of claims 1 to claim 6, wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell.

16. A kit comprising: a cell of claim 15, or a recombinant or synthetic nucleic acid of claim 13, or an expression vehicle of claim 14, or a synthetic, non- natural protein heterodimer of any of claims 1 to claim 6.

17. A biosensor or a microfluidic device comprising: a cell of claim 9, or a synthetic or non-natural protein complex of claim 7 or claim 8, or a synthetic, non- natural protein heterodimer of any of claims 1 to 6.

18. Use of a synthetic, non-natural protein heterodimer of any of the preceding claims to make a product of manufacture, wherein optionally the product of manufacture is or comprises a biomaterial, and optionally the biomaterial comprises a microfluidic device or a biosensor.

19. A synthetic, non-natural protein heterodimer of any of the preceding claims for use in making a product of manufacture, wherein optionally the product of manufacture is or comprises a biomaterial, and optionally the biomaterial comprises a microfluidic device or a biosensor.

20. An isolated or recombinant synthetic polypeptide comprising a sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDPATENT 5810.152599PCT / Love-J7 NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, optionally further comprising a moiety or amino acid sequence capable of binding to a detectable compound, composition or moiety, and optionally further comprising added to the C terminus (GGSGGCC) (SEQ ID NO:41).

21. A gold nanoparticle comprising an isolated or recombinant synthetic polypeptide having a sequence as set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and gold particle.

22. A recombinant or synthetic nucleic acid encoding a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:

40.

23. An expression vehicle comprising a recombinant or synthetic nucleic acid of claim 22, wherein optionally the expression vehicle is a viral particle, a recombinant virus, a baculovirus, a phage, a plasmid, a phagemid, a cosmid, a fosmid, a bacterial artificial chromosome, a viral DNA, or a P1-based artificial chromosomes.

24. A cell having contained therein, or comprising, a recombinant or synthetic nucleic acid of claim 22, or an expression vehicle of claim 23, or a synthetic polypeptide of claim 20, wherein optionally the cell is a prokaryotic or a eukaryotic cell, or a mammalian or a human cell.PATENT 5810.152599PCT / Love-J7 25. A kit comprising: a cell of claim 24, or a recombinant or synthetic nucleic acid of claim 22, or an expression vehicle of claim 23, or a synthetic polypeptide of claim 20.

26. A biosensor or a microfluidic device comprising: a cell of claim 24, or a recombinant or synthetic nucleic acid of claim 22, or an expression vehicle of claim 23, or a synthetic polypeptide of claim 20.

27. A method for isolating a rare earth metal (REM) or rare earth element (REE) (optionally neodymium, lanthanum, dysprosium, gadolinium) comprising: (a) adding to a solution, optionally an aqueous solution, a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, wherein the solution comprises a rare earth metal (REM) or rare earth element (REE), and (b) isolating the peptide-REM or peptide-REE.

28. The method of claim 27, wherein the REE comprises a lanthanide, scandium or yttrium.

29. The method of claim 28, wherein the lanthanide selected from the group consisting of: Lanthanum (La) - Atomic Number 57; Cerium (Ce) - Atomic Number 58; Praseodymium (Pr) - Atomic Number 59 ;Neodymium (Nd) - Atomic Number 60; Promethium (Pm) - Atomic Number 61; Samarium (Sm) - Atomic Number 62; Europium (Eu) - Atomic Number 63; Gadolinium (Gd) - Atomic Number 64; Terbium (Tb) - Atomic Number 65; Dysprosium (Dy) - Atomic Number 66; Holmium (Ho) - Atomic Number 67; Erbium (Er) - Atomic Number 68; Thulium (Tm) - Atomic Number 69; Ytterbium (Yb) - Atomic Number 70; and, Lutetium (Lu) - Atomic Number 71.

30. A method for isolating a compound or composition, wherein the compound or composition comprises: a metal or metal ion, optionally a heavy metal or a metal oxide, optionally lead (optionally lead oxide or dead dioxide or Lead(II) oxide), cadmium (optionally cadmium oxide or cadmium(II) oxide), mercuryPATENT 5810.152599PCT / Love-J7 (optionally mercury(II) oxide or mercuric oxide), zinc (optionally a zinc cation, and optionally the zinc cation comprises zinc sulfate), chromium (optionally a chromium ion), a rare earth element (REE), a drug or a small molecule, a per- or a polyfluoroalkyl substances (PFAS), a formaldehyde, paraformaldehyde or a polyoxymethylene, asbestos or anthophyllite, a pesticide optionally, glyphosate, Dichlorodiphenyltrichloroethane (DDT), 2,4-D (2,4-dichlorophenoxy acetic acid), aminopyralid, atrazine, or clopyralid, an organophosphate, optionally malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, ethion, trichlorfon, soman, sarin, tabun, VX, tribufos, a poison or a toxin, optionally a toxin associated with 1st, 2nd, and 3rdhand cigarette and cigar smoke, including nicotine, benzene, arsenic, formaldehyde, a petroleum-based toxin or pollutant, optionally a persistent organic pollutant (POP) or a halogenated organic compound, optionally: aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobutadiene (HCB), mirex, toxaphene, a polychlorinated biphenyl molecule (PCB), dioxin or a polychlorinated dibenzofuran, arsenic, selenium or a chromium or chromium compound, optionally a trivalent chromium (Cr(III)) ion, a human or a plant hormones, optionally a steroids, estrogen or testosterone, or a performance enhancing drug, a peptide, optionally erythropoietin (EPO), a steroid, optionally an anabolic steroid, a biomolecular marker, optionally a biomolecular marker associated with cancer, optionally Prostate Specific Antigen (PSA), CA15-3, CA27.29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, chromogranin, alpha fetoprotein (AFP), or vimentin,PATENT 5810.152599PCT / Love-J7 the method comprising: (a) adding to a solution, optionally an aqueous solution, the compound or composition, and (b) isolating the compound or composition.

31. The method of claim 30 and 3333, wherein the REE or compound or composition is isolated or removed from the solution the by use of a: High- Performance Liquid Chromatography (HPLC); Reverse-Phase HPLC (RP-HPLC); Reversed-Phase Chromatography (RPC); Ion-Exchange Chromatography (IEX); Size- Exclusion Chromatography (SEC) immunoaffinity column; Chiral Chromatography or a combination thereof.

32. Use of a peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for isolating a rare earth metal (REM) or rare earth element (REE) (optionally a lanthanide, or neodymium, lanthanum, dysprosium, gadolinium).

33. A peptide or polypeptide having a sequence as set forth in: SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and or SEQ ID NO:33, for use in isolating a rare earth metal (REM) or rare earth element (REE) (optionally a lanthanide, or neodymium, lanthanum, dysprosium, gadolinium).

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