Structure and stability of 3-alpha vs alpha/beta folds

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DESCRIPTION (provided by applicant): The major difficulty in deciphering the protein folding code is that folding information is diffuse. The aim of this proposal is to better understand how amino acid sequence specifies unique tertiary folds by reducing the folding problem to those amino acids which contain the most information toward specifying one fold versus another. To do this, Dr. Orban proposes to generate and study pairs of proteins with high sequence identity but different tertiary structures. He will study switching between alpha/beta structure and three a-helix bundle structure (3-a) as a function of amino acid sequence and characterize the energy separating the two conformations by a variety of biophysical methods. The IgG-binding domains of streptococcal protein G (GB) and staphylococcal protein A (AB) will be used as one pair. The albumin binding domain of protein G (GA) and GB will be used as the second pair. All three domains are of similar size (45-58 amino acids). AB and GA have 3-alpha folds and GB has an alpha/beta fold. Phage display selection methods will be used to induce a conformational switch between the 3-a structure and the alpha/beta structure, while maintaining the highest practicable level of sequence identity. The PI will then use the thermodynamic linkage between folding and IgG- or albumin-binding to quantitate the folding information content of each amino acid which differs in homologous pairs. A variety of biophysical techniques including, multidimensional NMR, will be used to explore structural and energetic properties of the sequence space between the homologous protein pairs. More complete knowledge concerning how primary sequence determines stable, unique protein folds should greatly advance the fields of protein engineering, protein structure prediction and de novo protein design. There also may be applications of this technology to create molecular switches based on the property of a protein to change conformations in response to a subtle external stimulus.