Method for development of a peptide building block useful for de novo protein design

a technology peptides, applied in the field of peptide building blocks useful for de novo protein design, can solve the problems of significant unresolved questions regarding the practical limitations of symmetric protein design, and achieve the effect of increasing the overall thermostability and the overall thermostability

Inactive Publication Date: 2011-09-15
FLORIDA STATE UNIV RES FOUND INC
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Benefits of technology

[0030]FIG. 10. SD transform #2: introduction of a symmetric constraint on reverse turn regions. Gray shading indicates positions related by 3-fold structural symmetry where either two or all three residues are identical. Boxed positions indicate the specific site(s) of mutation in construction of that particular mutant. ΔΔG values are a measure of the stability effect (referencing FGF-1), where a negative value indicates an increase in overall thermostability. Thermodynamic data are from Lee, J. & Blaber, M. (2011). Proc. Natl Acad. Sci. USA, 108, 126-130. FIG. 10 discloses SEQ ID NOS 15-18, respectively, in order of appearance.
[0031]FIG. 11. SD transform #3: introduction of a symmetric

Problems solved by technology

However, there are also significant unresolved questions reg

Method used

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  • Method for development of a peptide building block useful for de novo protein design
  • Method for development of a peptide building block useful for de novo protein design
  • Method for development of a peptide building block useful for de novo protein design

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example 1

Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif

[0066]The majority of protein architectures exhibit elements of structural symmetry, and “gene duplication and fusion” is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique “top-down symmetric deconstruction” strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed “conserved architecture” model for symmetric prote...

example 2

Supporting Information for Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif

[0082]Mutagenesis and Protein Purification. Construction of all the “SYMΔΔ” mutants utilized a synthetic gene for the 140 amino acid form of human fibroblast growth factor-1 (FGF-1) (Gimenez-Gallego et al. (1986) Biochem Biophys Res Commun 128:611-617, Linemeyer D L et al. (1990) Growth Factors 3:287-298, Ortega S et al. (1991) J Biol Chem. 266:5842-5846 and Blaber et al. (1996) Biochemistry 35:2086-2094) containing an additional amino-terminal six His tag (SEQ ID NO: 3) and following previously described procedures (Brych et al. (2001) Protein Sci 10:2587-2599). Construction of the Symfoil-1 (for symmetric β-trefoil protein 1) mutant involved complete gene synthesis utilizing unique codons at symmetry-related positions. Expression and purification of recombinant proteins followed previously published procedures (Brych et al. (2001) Protein Sci 10:2587-2599)...

example 3

A Polypeptide “Building Block” for the β-Trefoil Fold Identified by “Top-Down Symmetric Deconstruction”

[0089]Transform #1: Top-down SD of core positions. Attempts to introduce a symmetric constraint on the hydrophobic core-packing group, without also making alterations to the asymmetric FGF-1 tertiary structure, met with limited success. An alternative core-packing group involving five residue positions (SYM5 mutant, FIG. 8) was identified that was essentially equivalent to the FGF-1 thermostability and folding cooperativity (Brych, S. R. et al. (2001). Protein Sci. 10, 2587-2599 and Brych, S. R. et al. (2003) Protein Sci. 12, 2704-2718). Folding and unfolding kinetic data showed that the SYM5 mutant exhibited folding and unfolding rates similar to that of the FGF-1 protein including the characteristic biphasic folding behavior (Brych, S. R. et al. (2003) Protein Sci. 12, 2704-2718). Attempts at introducing a further symmetric constraint (Ile) at symmetry-related positions 25, 67, a...

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Abstract

The present invention relates to a top-down symmetric deconstruction approach which provides a novel alternative means to successfully identify a useful polypeptide “building block” for subsequent “bottom-up” de novo design of target protein architecture. The present invention also pertains to a novel peptides isolated by top-down symmetric deconstruction which may be useful for design or directed evolution of novel proteins with novel functionalities.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE[0001]This application claims priority to U.S. provisional patent application Ser. No. 61 / 312,843 filed Mar. 11, 2010. Reference is made to U.S. provisional patent application Ser. No. 61 / 179,751 entitled “Fibroblast Growth Factor Mutants Having Improved Functional Half Life and Methods of Their Use” filed May 20, 2009 and U.S. provisional patent application Ser. No. 61 / 309,590 entitled “Fibroblast Growth Factor Mutants Having Improved Functional Half Life and Methods of Their Use” filed Mar. 2, 2010.[0002]The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any pr...

Claims

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Application Information

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IPC IPC(8): C07K2/00G06G7/58G16B15/20G16B30/10
CPCC07K1/00G06F19/22G06F19/16G16B15/00G16B50/00G16B30/00G16B15/20G16B30/10C07K14/50
Inventor BLABER, MICHAELLEE, JIHUN
Owner FLORIDA STATE UNIV RES FOUND INC
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