Compositions and methods for homology-based derivation of structure-function relationships for protein families

Abstract

Compositions, systems, kits, and methods are disclosed that provide a new and improved strategy for studying structure-function relationships of functional properties of proteins, as well as methods of designing new proteins that possess multiple desired functional properties / property values. The structure-function rules derivable using this method are useful, for example, for both protein engineering and for improved functional annotation of genes. The methods involve (for example, but not by way of limitation) comparison of two existing homologs via multiple sequence alignments and phylogenetic trees to identify common ancestors that possessed both desired functional properties, along with other ancestors between the common ancestor and the current homologs. Synthetic genes encoding the predicted ancestral sequences are designed, and the genes are used to overproduce the predicted ancestral proteins for analysis of the desired functions. Analysis of the biochemical properties of the predicted ancestral proteins is utilized to identify where in the evolutionary path from last common ancestral proteins to present-day proteins the large changes in protein function occurred. The methods can also involve analysis of a single homolog that possesses or does not possess a functional property that is not shared by a common ancestor to identify a structure-function relationship for that functional property. In addition, based on these analyses, new synthetic proteins can be designed that possess one or more desired functions, or that do not possess one or more undesired functions. DNA sequences, vectors, and recombinant host cells encoding the synthetic proteins are also disclosed.

Description

COMPOSITIONS AND METHODS FOR HOMOLOGY-BASED DERIVATION OF STRUCTUREFUNCTION RELATIONSHIPS FOR PROTEIN FAMILIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 505,312, filed May 31, 2023. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via EFS-Web in computer readable form as an XML file. The Sequence Listing, created May 29, 2024, is named "57910.237wo SequenceListing.xml" and is 3,147 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference.BACKGROUND

[0003] A generally occurring challenge in the areas of protein science and protein engineering is that different members of a protein family have functionally distinct properties. Because these protein homologs often have substantially different amino acid sequences, there is a combinatorial explosion of an almost infinite number of different possible combinations of residue substitutions at the divergent residues that result in these differences in properties. Currently, it is generally not possible to determine which combination(s) of substitutions cause observed functional differences. In addition to being important as a general feature of protein science, this issue limits both functional precision in gene annotation (including annotation of medically important proteins) and protein engineering (including engineering of industrially important proteins).

[0004] The current methods for studying structure-function relationships involve site- directed mutagenesis, the comparison of homologs, and the production of chimeras. However, each of these methods has drawbacks and deficiencies. For example, site-directed mutagenesis only allows for the examination of one or two residues at a time. In addition, the comparison of homologs and production of chimeras can run into context dependency issues. Therefore, there is a need in the art for new and improved methods of studying structure-function relationshipsin proteins. It is to such methods, and synthetic compositions produced therefrom, that the present disclosure is directed.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 contains a flow chart illustrating one non-limiting embodiment of a method of studying structure-function relationships in accordance with the present disclosure. The result of this process generally yields two ancestral proteins that are very similar in amino acid sequence but quite different in one or more functional properties, resulting in the desired identification of one or more substitutions or combinations of substitutions that cause the observed functional difference.

[0006] FIG. 2 illustrates an ultra-high-resolution structure of a bacterial photoreceptor, Photoactive Yellow Protein (PYP) (upper panel), while the lower panel includes a simplified scheme of the light-triggered PYP photocycle and a structural model for the active site of PYP, highlighting major molecular events during its photocycle.

[0007] FIG. 3 illustrates a summary of the biochemical properties of the PYP family, including 11 existing PYP homologs that have been studied to date. In addition, a sequence alignment of a portion of Hhall (SEQ ID NO:1) and H hal 2 (SEQ ID NO:2) is provided.

[0008] FIG. 4 illustrates one non-limiting embodiment of an analysis of a set of resurrected ancestral sequences for PYP ancestors generated by the protein bioinformatics pipeline of the present disclosure.

[0009] FIG. 5 illustrates one non-limiting embodiment of the present disclosure's approach to analyzing resurrected ancestral sequences, using PYP as an example.

[0010] FIG. 6 graphically depicts one non-limiting embodiment of a construct used to overexpress ancestors (PYP Node 13) after they were synthetically designed.

[0011] FIG. 7 contains a photograph demonstrating the characteristic yellow color of the protein on an anion exchange column, demonstrating successful resurrection of an ancestor of Hhal PYP1 and Hhal PYP2 that is a functional PYP.DETAILED DESCRIPTION

[0012] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary language and results, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The inventive concept(s) is capable of other embodimentsor of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0013] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0014] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed inventive concept(s) pertains. All patents, published patent applications, and nonpatent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0015] All of the compositions and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the inventive concept(s) have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concept(s). All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the appended claims.

[0016] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0017] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaningof "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0018] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0019] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0020] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for exam pie," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0021] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of errorfor a composition / apparatus / device, the method beingemployed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, orfour percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0022] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0024] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0025] The term "isolated" as used herein means that a biological material, such as but not limited to a nucleic acid or protein, has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The same polynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being "isolated."

[0026] The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity andwhich may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients, and the like.

[0027] "Isolated polypeptide" or "isolated protein" as used herein means a polypeptide or protein which is substantially free of those compounds that are normally associated with the polypeptide or protein in a natural state, including but not limited to, other proteins or polypeptides, nucleic acids, carbohydrates, lipids and the like.

[0028] The term "purified" as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of the natural material, for example but not by way of limitation, two, three, four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term "purified" as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.

[0029] The term "variant" as used herein when referring to, for example, polynucleotides encoding a polypeptide variant of a given reference polypeptide, are polynucleotides that differ from the reference polypeptide but generally maintain their functional characteristics of the reference polypeptide. A variant of a polynucleotide may be a naturally occurring allelic variant or may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the reference polynucleotide can be made by, for example but by way of limitation, mutagenesis techniques, including those mutagenesis techniques that are applied to polynucleotides, cells, or organisms.

[0030] The term "homolog" as used herein will be understood to refer to proteins from present-day, currently living organisms that derive from a common ancestor. The term "homolog," when used alone to refer to a protein, is distinct from a "resurrected ancestral protein homolog," as otherwise described herein.

[0031] As used herein, the terms "nucleic acid segment," "nucleic acid sequence," "nucleotide segment," "nucleotide sequence," "DNA sequence," and "DNA segment" are used interchangeably and referto a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" or "isolated" nucleotide sequence as used herein refers to a DNA segment which contains a desired coding sequence yet is isolated away from, or purified free from, unrelated genomic DNA. Included within these terms are DNA segments and smaller fragments of such segments, and also recombinant vectors including, for example (but not by way of limitation), plasmids, cosmids, phage, viruses, and the like.

[0032] Similarly, a DNA segment comprising a desired gene refers to a DNA segment including coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein-, polypeptide-, or peptide-encoding unit. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, synthetic sequences, or combinations thereof. "Isolated substantially away from other coding sequences" means that the gene of interest forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or DNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to, or intentionally left in, the segment by the hand of man.

[0033] In certain embodiments, DNA sequences in accordance with the presently disclosed and claimed inventive concept(s) will further include genetic control regions which allow the expression of the sequence in a selected recombinant host. Of course, the nature of the control region employed will generally vary depending on the particular use (e.g., cloning host) envisioned.

[0034] The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects, such as (but not limited to) toxicity, irritation, and / or allergic response, commensurate with a reasonable benefit / risk ratio.

[0035] The term "patient" or "subject" as used herein includes human and veterinary subjects. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.

[0036] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition / disease / infection as well as individuals who are at risk of acquiring a particular condition / disease / infection (e.g., those needing prophylactic / preventative measures). The term "treating" refers to administering an agent to a subject / patient for therapeutic and / or prophylactic / preventative purposes.

[0037] A "therapeutic composition" or "pharmaceutical composition" refers to an agent that may be administered in vivo to bring about a therapeutic and / or prophylactic / preventative effect.

[0038] The terms "administration" and "administering," as used herein, will be understood to include all routes of administration known in the art. In addition, the compositions of the present disclosure (and / or the methods of administration of same) may be designed to provide delayed, controlled, or sustained release using formulation techniques which are well known in the art.

[0039] The term "pharmaceutically acceptable carrier or excipient" includes any carriers or excipients known in the art may be utilized in accordance with the present disclosure. For example (but not by way of limitation), a physiological compatible carrier (e.g., saline) that is compatible with maintaining the structure / activity of the active ingredient(s) when administered, and compatible with the desired mode of administration, may be utilized as the pharmaceutically acceptable carrier in accordance with the present disclosure. In addition, the active ingredient(s) may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient(s). Suitable excipients include, for example but not byway of limitation, water, saline, dextrose, glycerol, ethanol, and the like, or any combination thereof.

[0040] Circuitry, as used herein, may be analog and / or digital components, or one or more suitably programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. Also, "components" may perform one or more functions. The term "component," may include hardware, such as a processor (e.g., microprocessor), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a combination of hardware and software, and / or the like. The term "processor" as used herein means a single processor or multiple processors working independently or together to collectively perform a task.

[0041] Software may include one or more computer readable instructions that when executed by one or more components cause the component to perform a specified function. It should be understood that the algorithms described herein may be stored on one or more non- transitory computer readable medium. Exemplary non-transitory computer readable mediums may include random access memory, read only memory, flash memory, and / or the like. Such non-transitory computer readable mediums may be electrically based, optically based, magnetically based, and / or the like. Further, the messages described herein may be generated by the components and result in various physical transformations.

[0042] Turning now to the inventive concepts, methods of creating and employing bioinformatics analyses of protein families (including, but not limited to, high quality sequence alignments, high quality phylogenetic trees, and / or ancestral protein predictions) are utilized todetermine structure-function relationships. The structure-function rules derivable using this method are useful, for example, for both protein engineering and for improved functional annotation of genes. The methods involve the prediction of amino acid sequences of ancestral proteins as well as the creation of synthetic gene sequences encoding same. These synthetic gene sequences are then utilized to produce "resurrected ancestral proteins" and determine the activities and functions thereof. Comparison of one or more existing homologs in the protein family to one or more resurrected ancestral proteins, as well as comparison of properties of two or more resurrected ancestral proteins, is utilized to identify changes in amino acid sequence that define a structure-function relationship. The methods of the present disclosure allow for the production of designer proteins that possess specific functional properties and specific selected combinations of functional properties.

[0043] Certain non-limiting embodiments of the present disclosure are directed to a method of determining at least one structure-function relationship for at least one functional property in a family of proteins, wherein a substantial difference in the value of the at least one functional property is observed for two current-day homologs in the family of proteins. The method includes the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) at last common ancestral protein of both the first and second homologs; (b) at least a first additional ancestral protein between (a) and the first homolog; and (c) at least a second additional ancestral protein between (b) and the first homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the functional property; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and (10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid thataffected the value of the functional property.

[0044] Certain non-limiting embodiments of the present disclosure are directed to a method of determining at least one structure-function relationship for at least one functional property in a family of proteins, wherein a substantial difference in the value of the at least one functional property is observed for two current-day homologs in the family of proteins. The method includes the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) at last common ancestral protein of both the first and second homologs; and (b) at least a first additional ancestral protein between (a) and the first homolog; and (c) at least a second additional ancestral protein between (b) and the first homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the functional property; (8A) optionally identifying (d) at least a third additional ancestral protein between (a) and (b), (b) and (c), or (c) and the first homolog; (8B) preparing a synthetic DNA sequence encoding the predicted amino acid sequence of (d), producing the ancestral protein (d) in a suitable host organism, and assaying the ancestral protein (d) to determine a value for the functional property; (8C) optionally repeating steps (8A) and (8B) until a difference in the value of the functional property from the functional property value of the first homolog is observed; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and (10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid that affected the value of the functional property.

[0045] Certain non-limiting embodiments of the present disclosure are directed to a method of determining at least one structure-function relationship for at least two functional properties in a family of proteins, wherein a substantial difference in the value of the at least one functionalproperty is observed for two current-day homologs in the family of proteins. The method includes the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for a first functional property and a value for a second functional property, and a second homolog possesses values for the first and second functional properties that are different from the values of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first homolog, the second homolog, and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) a last common ancestral protein of both the first homolog and the second homolog; ( b) at least one additional ancestral protein between (a) and the first homolog; and (c) at least one additional ancestral protein between (a) and the second homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the first functional property and a value for the second functional property; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property, and / or identifying a region in the reconstructed evolutionary path from (a) to the second homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property; and (10) determining at least a portion of an amino acid sequence associated with the first functional property and / or the second functional property based on the change in the at least one amino acid that affected the value of said functional property.

[0046] In certain particular (but non-limiting) embodiments of the above-described methods, steps (5)(b), (5)(c), (6), (7), and (8) (and / or steps (8A), (8B), and (8C), when present) may be repeated one or more times until steps (9) and (10) can be performed. Repeating these steps and analyzing more ancestral proteins allows the method to "zoom in" on a selected region of the reconstructed evolutionary pathway to identify the "minimal segment" of that pathway that results in changes in a functional property (such as, but not limited to, rate or stability). In the general case, this "minimal segment" then tremendously reduced the number of substitutions between the two homologs adjacent in this minimal segment, yielding the most informative insights into the structure-function relationships for the selected functionalproperty.

[0047] The first functional property (and second functional property, when present) may be any functional property known in the art or otherwise disclosed herein. Non-limiting examples include a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof. Note that the differences in functional properties do not require the presence or absence of a functional property; rather, a first functional property may be a slower or faster stability, catalytic, or binding rate than the second functional property. For example (but not by way of limitation), a first functional property may be a slower catalytic rate, and the second functional property may be a faster catalytic rate.

[0048] Certain non-limiting embodiments of the present disclosure include synthetic proteins or peptides containing one or more amino acid differences from an existing, current- day homolog, wherein the one or more amino acid differences is determined by the methods disclosed or otherwise contemplated herein. In one non-limiting example, the synthetic protein comprises a desired value for a first functional property. In another non-limiting example, the synthetic protein comprises a desired value for a first functional property and a desired value for a second functional property, as well as at least one amino acid that is different from either the first or second homolog.

[0049] Certain non-limiting embodiments of the present disclosure also include isolated, synthetic DNA sequences that encode any of the synthetic proteins disclosed or otherwise contemplated herein. The synthetic nucleotide sequences comprise at least one nucleotide that differs from a native sequence and thus is a non-natu rally occurring nucleotide sequence.

[0050] Certain non-limiting embodiments of the present disclosure also include vectors that comprise any of the isolated DNA sequences disclosed or otherwise contemplated herein. In a particular (but non-limiting) embodiment, the vector further comprises at least one genetic control region that allows for expression of the DNA sequence.

[0051] Certain non-limiting embodiments of the present disclosure also include recombinant host cells that include any of the isolated DNA sequences and / or any of the vectors disclosed or otherwise contemplated herein.

[0052] Certain non-limiting embodiments of the present disclosure are directed to kits or systems for performing a portion or all of any of the methods disclosed or otherwise contemplated herein. The kits or systems may further include one or more components for producing one or more of any of the isolated DNA sequences, vectors, recombinant host cells, or synthetic proteins disclosed or otherwise contemplated herein.

[0053] Certain non-limiting embodiments of the present disclosure are directed to a pharmaceutical composition that comprises one or more of any of the recombinantly produced synthetic peptides or proteins disclosed or otherwise contemplated herein in combination with at least one pharmaceutically acceptable carrier.

[0054] Certain non-limiting embodiments of the present disclosure are directed to a method that comprises administering one or more of any of the pharmaceutical compositions disclosed or otherwise contemplated herein to a subject in need thereof.EXAMPLES

[0055] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.Example 1

[0056] A generally occurring challenge in the areas of protein science and protein engineering is that different members of a protein family have functionally distinct properties. Because these protein homologs often have substantially different amino acid sequences, because of the combinatorial explosion of many different possible combinations of residue substitutions, currently in general it is not possible to determine which substitutions cause observed functional differences. In addition to being important as a general feature of protein science, this issue limits both functional precision in gene annotation (including annotation of medically important proteins) and protein engineering (including engineering of industrially important proteins). Regarding protein engineering, it can be observed that two members of a protein family each have a property that is helpful for a specific industrial application. For example, one protein homolog can exhibit high thermal stability, while the other homolog exhibits a high catalytic rate. The approach reported here allows an avenue to identify the residues needed for these two properties, allowing the engineering of proteins that combine the two helpful properties. In the approach of the present disclosure, the availability of large amounts of DNA sequence information is used, and many homologs of a protein under study are first identified. Then this information is used to predict all ancestral proteins that gave rise to the observed sequence variation in this protein family. By studying the biochemical properties of strategically selected ancestral proteins after their resurrection based on synthetic genetechnology, the substitutions that are responsible for specific functional differences are rapidly and effectively identified. Therefore, this approach provides an effective and generally implementable tool for addressing the challenge of the combinatorial explosion of substitutions to identify structure-function rules in protein families.

[0057] The process that forms the basis of certain non-limiting embodiments of the present disclosure is as follows:

[0058] (1) Select a protein of interest for which (i) there is a sufficiently large number of homologs belonging to the same protein family available in sequence databases; and (ii) for which information is available on functional differences of interest as exhibited by different members of this protein family. The method reported here works best for proteins that can be overproduced sufficiently well and for which sufficiently convenient and quantitative functional essays are available.

[0059] (2) Identify (and manually curate as needed) all protein homologs belonging to the same protein family as the protein of interest and generate a high-quality multiple sequence alignment (MSA) of the amino acid sequences of these homologs.

[0060] (3) Use this MSA to compute a high-quality phylogenetic tree of these proteins and use this tree to compute high quality predictions for the amino acid sequences of all of the protein ancestors that gave rise to the observed sequence variation in the protein family of interest. It should be noted that if the number of proteins in the protein family becomes large (i.e., over 1,000), then substantial computational resources are needed to compute the set of ancestral protein sequences.

[0061] (4) Identify where in the phylogenetic tree the various proteins with the helpful properties of interest are located. Identify the last common ancestor of pairs of proteins with helpful properties and a small number of strategically located ancestral proteins in between this last common ancestor and the present-day proteins of interest. Design synthetic DNA sequences encoding this set of predicted ancestral sequences, obtain the corresponding synthetic genes (such as, but not limited to, by ordering from available commercial vendors), clone these genes in suitable vectors, and overproduce the predicted ancestral proteins in a suitable host organism, such as (but not limited to) E. coli. Measure the biochemical properties of these resurrected ancestral proteins to identify where in the evolutionary path from last common ancestral proteins to present-day protein large changes in protein function occurred.

[0062] (5) Based on this information, select a next set of predicted ancestral proteins to further narrow down where in the evolutionary pathway the changes in functional propertiesoccurred. This is a variant of a binary search algorithm, and thus will very efficiently (in a relatively small number of iterations) identify sets of predicted ancestral proteins that are quite similar in amino acid sequence but quite different in functional properties, thereby revealing which residues cause these functional differences.

[0063] The section of the phylogenetic tree of the protein family to which the derived structure-function rules apply can be efficiently determined. In a favorable case, these rules will apply both to the last common ancestor and the pair of present-day proteins being studied. Otherwise, additional iterations of this approach can be performed to obtain the additional structure-function rules involved. The derived structure-function rules are then utilized to engineer proteins that combine the favorable properties of the pair of protein homologs identified at the start of the project.

[0064] In many cases, the entire above procedure is open to the application of automation and high-throughput methods of cloning, optional protein purification, and 96-well based functional essays, greatly accelerating the process of discovering the needed structure-function relationships.

[0065] FIG. 1 contains a flow chart illustrating one non-limiting embodiment of the method of the present disclosure that includes two stages. Stage 1 includes a general purpose pipeline for bioinformatics analysis of protein families, while Stage 2 analyzes the biochemistry of resurrected ancestral proteins.

[0066] In particular, Stage 1 includes the use of NCBI's BlastP alignment search tool to identify a sufficiently large number of homologs belonging to the same protein family as a protein of interest and that are available in sequence databases and for which information is available on functional differences of interest. Then a high-quality multiple sequence alignment (MSA) of the amino acid sequences of these homologs is generated. The MSA is used to compute a high-quality phylogenetic tree of these proteins, and this tree is used to compute high quality predictions for the amino acid sequences of all of the protein ancestors that gave rise to the observed sequence variation in the protein family of interest. Based on the phylogenetic tree, the last common ancestor of pairs of proteins with helpful properties and a small number of strategically located ancestral proteins in between this last common ancestor and the present- day proteins of interest can be identified. In Stage 2, synthetic DNA sequences encoding this set of predicted ancestral sequences are designed, cloned into suitable vectors, and overproduced in a suitable host organism. Then the biochemical properties of these resurrected ancestral proteins are measured, either in a whole-cell assay or in a cell-free assay following purificationof each of the predicted ancestral proteins from the suitable host organism. For example, but not by way of limitation, a whole-cell assay may be utilized when the protein family of interest is an enzyme that causes the cell to become colored or fluorescent, either because the protein is colored or because the protein is a transcription factor controlling for example the expression of a gene encoding a colored or fluorescent protein (such as, but not limited to, green fluorescent protein). Alternatively, the ancestral proteins may be isolated and purified away from the suitable host organism and then subjected to commonly used protein assays.

[0067] Based on the measured biochemical properties of the resurrected ancestral proteins, it is identified where in the evolutionary path from last common ancestral proteins to present- day protein large changes in protein function occurred, thereby allowing for the identification of residues that cause desired functional differences.

[0068] Currently, there is no available method to address the generally occurring problem of the "combinatorial explosion" in identifying family-wide protein structure-function rules to engineer proteins that combine beneficial properties observed in different homologs of a protein family into a single protein. The present disclosure offers an approach to uncover protein structure-function relationships for use in the pharmaceutical industry, national laboratories, and academics and for protein engineering to help industrial partners address their specific protein family structure-function needs.

[0069] As described in Example 2, this novel method has been applied to the model protein family of photoactive yellow protein. This case exhibits all of the complexities of the barrier that the present disclosure overcomes, and thus establishes certain non-limiting embodiments of the disclosed approach. This approach promises to be helpful in some pharmaceutical applications, in improving the precision of genome annotations of selected protein families, and in engineering enzymes with industrial applications.Example 2

[0070] This Example illustrates structure-function studies conducted using ancestral sequence reconstruction of photoactive yellow protein as a model system, in accordance with the methods of the present disclosure.

[0071] The photoactive yellow protein (PYP) is a small, water-soluble protein, first identified in Halorhodospira halophila. PYP is a bacterial photoreceptor that has emerged as a powerful model system for protein biophysics. As demonstrated in FIG. 2, PYP exhibits a light-triggered photocycle that proceeds in three basic steps. First, the chromophore is photo-isomerized. Next,proton transfer occurs, triggering large structural changes of the protein. Lastly, thermal reisomerization and resetting of the initial protonation states occurs, which cause the protein to re-fold and to recover the initial ground state.

[0072] The present Example utilizes the approach of the present disclosure based on reconstructing ancestral proteins of PYPs to identify otherwise intractable structure-function relationships. This approach is based on ancestral sequence reconstruction; reconstruction of ancestral PYP sequences requires a large set of PYP homologs and a high-quality multiple sequence alignment. The approach was conducted as shown in FIG. 1.

[0073] As shown in FIG. 3, 11 PYPs in total have been studied experimentally to determine the speed of the recovery of the initial state of PYP. Some PYPs have a recovery rate of 0.5 seconds, while other PYPs have recovery rates ranging from 50 seconds to 60 minutes (see boxed column in FIG. 3 for specific values). Hhal PYP1 and Hhal PYP2 (circles) were chosen as the focus of this Example because they show a large difference in functional property (photocycle rate) while being the closest in sequence identity. These two proteins have a sequence identity of 60%, which means that there are substitutions at 49 positions. As can be seen in the lower panel of FIG. 3, residues that are identical between Hhal PYP1 and Hhal PYP2 are shaded, illustrating the quite large number of substitutions between these two homologs of photoactive yellow protein. As such, it is important to determine which of these many changes / substitutions in amino acid sequence between these two homologs caused the observed change in the photocycle rate. This Example determines which residues cause this change in photocycle recovery rate by using an ancestral protein triangulation approach. The method proposed here is the first strategy to be able to address that question, and in a generally applicable way.

[0074] 998 PYP homologs were identified for which a high-quality multiple sequence alignment could be obtained. This alignment allowed us to predict the amino acid sequences of all 996 nodes in the phylogenetic tree of these PYPs.

[0075] FIG. 4 illustrates the analysis of the set of resurrected ancestral sequences for PYP ancestors generated by the protein informatics pipeline of the present disclosure. This maximum likelihood tree was generated using the online tool i-TOL (European Molecular Biology Laboratory, Heidelberg, DE). 998 manually verified PYP homologs were identified. After a high- quality MSA was generated, a PYP phylogenetic tree was created in order to predict the ancestral sequences. IQ-TREE software was used to predict the ancestral sequences. The amino acid sequences of 996 ancestors (nodes) were predicted that are available for generic and biochemical resurrection. Sequences selected for resurrection were first inspected for unusualsubstitutions, and their predicted 3D structure was checked using RobettaFold (Baker Lab, University of Washington).

[0076] The approach for analyzing the resurrected ancestral sequences is shown in FIG. 5. The strategy for this analysis was to identify where in this pathway large changes in photocycle rate occurred.

[0077] Following this analysis, an ancestor of interest (PYP Node 13) was selected, and a synthetic pyp gene encoding the predicted sequence of PYP Node 13 was designed. The synthetically designed pyp gene was then inserted into the construct of FIG. 6, and the construct was used to overexpress the ancestor for biochemical and spectroscopic analysis of its functional rate. As shown in FIG. 7, the protein expressed from the construct exhibited the characteristic yellow color of PYP proteins on an anion exchange column, thus demonstrating successful resurrection of an ancestor of Hhal PYP1 and Hhal PYP2 that yielded a functional PYP.

[0078] The strategy utilized in the methods disclosed herein (and as depicted in FIG. 5) is to identify a "region" in the reconstructed evolutionary path where two proteins "immediately adjacent" in the reconstructed evolutionary path have substantially different functional properties (such as slow versus fast rates of a functional property). Because these two ancestral proteins are "immediately adjacent" in the evolutionary path, their amino acid sequences are quite similar to one another, and are much more similar to one another than to the initial selected two present-day homologs. When such a region in the reconstructed evolutionary path is identified, one can order more "finely spaced" reconstructed ancestors in this section of the evolutionary path to identify the "minimal" region in the evolutionary path where the functional change (for example from slow to fast rates of a functional property) occurred. It may well be that the two closely related resurrected ancestors thus identified have multiple substitutions in their sequence. Traditional site-directed mutagenesis methods can then be used if wanted / needed to check if indeed all of these substitutions are required for the observed functional change.

[0079] Therefore, the Examples demonstrate that the ancestral sequence reconstruction methods of the present disclosure provide a new, fourth approach for protein structure-function analysis, complementing (as well as providing advantages over) site-directed mutagenesis and studies on homologs and chimeras.

[0080] The protein family analysis pipeline of this Example identified 331 novel PYPs (many with interesting features) and yielded high-quality predicted sequences of ancestral PYPs. In addition, this Example demonstrated the successful resurrection of the first predicted ancestralPYP.

[0081] Additional embodiments of this Example include cloning genes encoding a series of strategically selected resurrected ancestral PYPs into E. coli, and then expressing these proteins and measuring their biochemical properties (either in a whole-cell assay or in a cell-free assay following isolation and purification of each of the predicted ancestral proteins). These steps can be iterated a small number of times to derive (and then test) structure-function rules for functional properties in (part of) the PYP family.Example 3

[0082] In another Example, two functional properties can be examined together to produce designer proteins with two desired functional properties. That is, the method starts with one present-day homolog that has a fast catalytic rate but that is unstable, and a second present-day homolog that has a slow catalytic rate but is stable for a desired period of time. The methods disclosed herein can generate a synthetic protein that combines the best of both functional properties and has both a fast catalytic rate as well as a desired stability. In order to produce the synthetic protein, the structure-function rules for one desired property is determined, and then changes are made in the protein with the other desirable property to produce a protein possessing both desired proteins. For example, the method may be designed to discover the structure-function rules that make a protein possess a fast catalytic rate. The changes can be made to the amino acid sequence of the "slow but stable" protein homolog to produce a synthetic protein with both a fast catalytic rate and a desired stability. Alternatively, the method may be designed to discoverthe structure-function rules that make a protein stable, and changes made to the amino acid sequence of the "fast but unstable" protein homolog to produce the synthetic protein with both fast catalytic rate and desired stability.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0083] Illustrative embodiment 1. A method of determining at least one structure-function relationship for at least one functional property for a family of proteins, the method comprising the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family,wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) at last common ancestral protein of both the first and second homologs; (b) at least a first additional ancestral protein between (a) and the first homolog; and (c) at least a second additional ancestral protein between (b) and the first homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), ( b), and (c) to determine a value for the functional property; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and (10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid that affected the value of the functional property.

[0084] Illustrative embodiment 2. The method of Illustrative embodiment 1, further comprising repeating steps (5)(b), (5)(c), (6), (7), and (8) one or more times until steps (9) and (10) can be performed.

[0085] Illustrative embodiment 3. The method of Illustrative embodiment 1 or 2, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

[0086] Illustrative embodiment 4. A synthetic protein, comprising at least one amino acid that is different from the first homolog, wherein the at least one amino acid difference is determined by the method of any of Illustrative embodiments 1-3, and wherein the synthetic protein possesses a desired value for the functional property.

[0087] Illustrative embodiment 5. An isolated DNA sequence encoding the synthetic protein of Illustrative embodiment 4.

[0088] Illustrative embodiment 6. A vector comprising: the isolated DNA sequence of Illustrative embodiment 5; and at least one genetic control region that allows for expression of the DNA sequence.

[0089] Illustrative embodiment 6A. A recombinant host cell comprising the isolated DNA sequence of Illustrative embodiment 5 and / or the vector of Illustrative embodiment 6.

[0090] Illustrative embodiment 7. A method of determining at least one structure-functionrelationship for at least two functional properties for a family of proteins, the method comprising the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for a first functional property and a value for a second functional property, and a second homolog possesses values for the first and second functional properties that are different from the values of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first homolog, the second homolog, and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) a last common ancestral protein of both the first homolog and the second homolog; ( b) at least one additional ancestral protein between (a) and the first homolog; and (c) at least one additional ancestral protein between (a) and the second homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the first functional property and a value for the second functional property; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property, and / or identifying a region in the reconstructed evolutionary path from (a) to the second homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property; and (10) determining at least a portion of an amino acid sequence associated with the first functional property and / or the second functional property based on the change in the at least one amino acid that affected the value of said functional property.

[0091] Illustrative embodiment 8. The method of Illustrative embodiment 7, further comprising repeating steps (5)(b), (5)(c), (6), (7), and (8) one or more times until steps (9) and (10) can be performed.

[0092] Illustrative embodiment 9. The method of Illustrative embodiment 7 or 8, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

[0093] Illustrative embodiment 10. The method of any of Illustrative embodiments 7-9, wherein the second functional property is selected from the group consisting of a specificstability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

[0094] Illustrative embodiment 11. A synthetic protein comprising: a desired value for the first functional property; a desired value for the second functional property; and at least one amino acid that is different from the first or second homolog; and wherein the at least one amino acid difference is determined by the method of any of Illustrative embodiments 7-10.

[0095] Illustrative embodiment 12. An isolated DNA sequence encoding the synthetic protein of Illustrative embodiment 11.

[0096] Illustrative embodiment 13. A vector comprising: the isolated DNA sequence of Illustrative embodiment 12; and at least one genetic control region that allows for expression of the DNA sequence.

[0097] Illustrative embodiment 13A. A recombinant host cell comprising the isolated DNA sequence of Illustrative embodiment 12 and / or the vector of Illustrative embodiment 13.

[0098] Illustrative embodiment 14. A method of determining at least one structure-function relationship for at least one functional property for a family of proteins, the method comprising the steps of: (1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog; (2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family; (3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors; (4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment; (5) identifying at least: (a) at last common ancestral protein of both the first and second homologs; and (b) at least a first additional ancestral protein between (a) and the first homolog; and (c) at least a second additional ancestral protein between (b) and the first homolog; (6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c); (7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism; (8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the functional property; (8A) optionally identifying (d) at least a third additional ancestral protein between (a) and (b), (b) and (c), or (c) and the first homolog; (8B) preparing a synthetic DNA sequence encoding the predicted amino acid sequence of (d), producing the ancestral protein (d) in a suitable host organism, and assaying the ancestral protein (d) to determine a value for thefunctional property; (8C) optionally repeating steps (8A) and (8B) until a difference in the value of the functional property from the functional property value of the first homolog is observed; (9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and (10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid that affected the value of the functional property.

[0099] Illustrative embodiment 15. The method of Illustrative embodiment 14, further comprising repeating steps (8A), (8B), and (8C) one or more times until steps (9) and (10) can be performed.

[0100] Illustrative embodiment 16. The method of Illustrative embodiment 14, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

[0101] Illustrative embodiment 17. A synthetic protein, comprising at least one amino acid that is different from the first homolog, wherein the at least one amino acid difference is determined by the method of any of Illustrative embodiments 14-16, and wherein the synthetic protein possesses a desired value for the functional property.

[0102] Illustrative embodiment 18. An isolated DNA sequence encoding the synthetic protein of Illustrative embodiment 17.

[0103] Illustrative embodiment 19. A vector comprising: the isolated DNA sequence of Illustrative embodiment 18; and at least one genetic control region that allows for expression of the DNA sequence.

[0104] Illustrative embodiment 19A. A recombinant host cell comprising the isolated DNA sequence of Illustrative embodiment 18 and / or the vector of Illustrative embodiment 19.

[0105] Illustrative embodiment 20. A pharmaceutical composition, comprising: one or more synthetic proteins of any of Illustrative embodiments 4, 11, and / or 17; and at least one pharmaceutically acceptable carrier.

[0106] Illustrative embodiment 21. A method, comprising: administering at least one pharmaceutical composition of Illustrative embodiment 20 to a subject in need thereof.

[0107] Illustrative embodiment 22. A kit or system for performing a portion or all of the method of any of Illustrative embodiments 1-3, 7-10, or 14-16.

[0108] Illustrative embodiment 23. A kit or system for producing at least one of: the synthetic protein of any of Illustrative embodiments 4, 11, or 17; the DNA sequence of any of Illustrativeembodiments 5, 12, or 18; the vector of any of Illustrative embodiments 6, 13, or 19; or the recombinant host cell of any of Illustrative embodiments 6A, 13A, or 19A.

[0109] Thus, in accordance with the present disclosure, there have been provided compounds, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

What is claimed is:

1. A method of determining at least one structure-function relationship for at least one functional property for a family of proteins, the method comprising the steps of:(1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog;(2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family;(3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors;(4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment;(5) identifying at least:(a) at last common ancestral protein of both the first and second homologs; and(b) at least a first additional ancestral protein between (a) and the first homolog; and(c) at least a second additional ancestral protein between (b) and the first homolog;(6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c);(7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism;(8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the functional property;(9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and(10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid that affected the value of the functional property.

2. The method of claim 1, further comprising repeating steps (5)(b), (5)(c), (6), (7), and (8) one or more times until steps (9) and (10) can be performed.

3. The method of claim 1, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

4. A synthetic protein, comprising at least one amino acid that is different from the first homolog, wherein the at least one amino acid difference is determined by the method of any of claims 1-3, and wherein the synthetic protein possesses a desired value for the functional property.

5. An isolated DNA sequence encoding the synthetic protein of claim 4.

6. A vector comprising the isolated DNA sequence of claim 5.

7. A method of determining at least one structure-function relationship for at least two functional properties for a family of proteins, the method comprising the steps of:(1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for a first functional property and a value for a second functional property, and a second homolog possesses values for the first and second functional properties that are different from the values of the first homolog;(2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family;(3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first homolog, the second homolog, and multiple protein ancestors;(4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment;(5) identifying at least:(a) a last common ancestral protein of both the first homolog and the second homolog;(b) at least one additional ancestral protein between (a) and the first homolog; and(c) at least one additional ancestral protein between (a) and the second homolog;(6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c);(7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism;(8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the first functional property and a value for the second functional property;(9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property, and / or identifying a region in the reconstructed evolutionary path from (a) to the second homolog where a change in at least one amino acid occurred that affected the value of the first or second functional property; and(10) determining at least a portion of an amino acid sequence associated with the first functional property and / or the second functional property based on the change in the at least one amino acid that affected the value of said functional property.

8. The method of claim 7, further comprising repeating steps (5)(b), (5)(c), (6), (7), and (8) one or more times until steps (9) and (10) can be performed.

9. The method of claim 7, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

10. The method of claim 7, wherein the second functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

11. A synthetic protein comprising: a desired value for the first functional property; a desired value for the second functional property; and at least one amino acid that is different from the first or second homolog; and wherein the at least one amino acid difference is determined by the method of any of claims 7-10.

12. An isolated DNA sequence encoding the synthetic protein of claim 11.

13. A vector comprising the isolated DNA sequence of claim 12.

14. A method of determining at least one structure-function relationship for at least one functional property for a family of proteins, the method comprising the steps of:(1) identifying a protein family comprising multiple homologs, wherein a first homolog possesses a value for the functional property and a second homolog possess a value for the functional property that is different from the value of the first homolog;(2) generating a multiple sequence alignment of amino acid sequences of the multiple homologs in the protein family;(3) generating a phylogenetic tree for the multiple homologs in the protein family, wherein the phylogenetic tree comprises the first and second homologs and multiple protein ancestors;(4) generating predicted amino acid sequences of protein ancestors that gave rise to observed sequence variation in the multiple sequence alignment;(5) identifying at least:(a) at last common ancestral protein of both the first and second homologs; and(b) at least a first additional ancestral protein between (a) and the first homolog; and(c) at least a second additional ancestral protein between (b) and the first homolog;(6) preparing synthetic DNA sequences encoding the predicted amino acid sequences of the ancestral proteins (a), (b), and (c);(7) producing each of the ancestral proteins (a), (b), and (c) in a suitable host organism;(8) assaying each of the ancestral proteins (a), (b), and (c) to determine a value for the functional property;(8A) optionally identifying (d) at least a third additional ancestral protein between (a) and (b), (b) and (c), or (c) and the first homolog;(8B) preparing a synthetic DNA sequence encodingthe predicted amino acid sequence of (d), producing the ancestral protein (d) in a suitable host organism, and assaying the ancestral protein (d) to determine a value for the functional property;(8C) optionally repeating steps (8A) and (8B) until a difference in the value of the functional property from the functional property value of the first homolog is observed;(9) identifying a region in a reconstructed evolutionary path from (a) to the first homolog where a change in at least one amino acid occurred that affected the value of the functional property; and(10) determining at least a portion of an amino acid sequence associated with the functional property based on the change in the at least one amino acid that affected the value of the functional property.

15. The method of claim 14, further comprising repeating steps (8A), (8B), and (8C) one or more times until steps (9) and (10) can be performed.

16. The method of claim 14, wherein the first functional property is selected from the group consisting of a specific stability rate, a specific catalytic rate, a specific binding characteristic, a specific binding rate, and combinations thereof.

17. A synthetic protein, comprising at least one amino acid that is different from the first homolog, wherein the at least one amino acid difference is determined by the method of any of claims 14-16, and wherein the synthetic protein possesses a desired value for the functional property.

18. An isolated DNA sequence encoding the synthetic protein of claim 17.

19. A vector comprising the isolated DNA sequence of claim 18.

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