Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code.
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.
Alexander
Patrick A
PA
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Rozak
David A
DA
Orban
John
J
Bryan
Philip N
PN
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D007074:Immunoglobulin G / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013816:Thermodynamics
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D019151:Peptide Library; D013205:Staphylococcal Protein A