Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code.

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TitleDirected evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code.
Publication TypeJournal Article
Year of Publication2005
AuthorsAlexander, PA, Rozak, DA, Orban, J, Bryan, PN
JournalBiochemistry
Volume44
Issue43
Pagination14045-54
Date Published2005 Nov 1
ISSN0006-2960
KeywordsBacterial Proteins, Binding Sites, Immunoglobulin G, Magnetic Resonance Spectroscopy, Mutagenesis, Site-Directed, Peptide Library, Protein Folding, Protein Structure, Secondary, Staphylococcal Protein A, Thermodynamics
Abstract

To better understand how amino acid sequences specify unique tertiary folds, we have used random mutagenesis and phage display selection to evolve proteins with a high degree of sequence identity but different tertiary structures (homologous heteromorphs). The starting proteins in this evolutionary process were the IgG binding domains of streptococcal protein G (G(B)) and staphylococcal protein A (A(B)). These nonhomologous domains are similar in size and function but have different folds. G(B) has an alpha/beta fold, and A(B) is a three-helix bundle (3-alpha). IgG binding function is used to select for mutant proteins which retain the correct tertiary structure as the level of sequence identity is increased. A detailed thermodynamic analysis of the folding reactions and binding reactions for a pair of homologous heteromorphs (59% identical) is presented. High-resolution NMR structures of the pair are presented by He et al. [(2005) Biochemistry 44, 14055-14061]. Because the homologous but heteromorphic proteins are identical at most positions in their sequence, their essential folding signals must reside in the positions of nonidentity. Further, the thermodynamic linkage between folding and binding is used to assess the propensity of one sequence to adopt two unique folds.

DOI10.1021/bi051231r
Alternate JournalBiochemistry
PubMed ID16245920
Grant ListGM62154 / GM / NIGMS NIH HHS / United States