The major outstanding question in structural biology is the protein folding problem. Rules for the acquisition of the final three- dimensional structure of most proteins are encoded in the information in the primary sequence of amino acids, however, a precise description of these rules is lacking. One popular model for protein folding is the hierarchical model in which the formation of the tertiary structure of a protein is realized through the packing of elements of secondary structure including alpha-helices and beta-sheets. Although this model is appealing in its simplicity, a direct test of the model has not been demonstrated. The study of alpha-helix formation in model peptides has provided a wealth of information on the contributions of specific side-chain interactions to the energetics of structure formation and a measure of the intrinsic helix-forming tendencies of the amino acids. The applicability of this information to protein stability, or even to helix formation in peptides derived from native proteins, has only been assumed and a direct test of the rules has not been performed. The availability of two related model protein systems, coupled with studies on isolated helical peptides from those proteins, will provide the first direct test of the hierarchical model for protein stability. The HPr protein from bacteria is an ideal vehicle for those studies since the proteins from both Escherichia coli and Bacillus subtilis are available in sufficient quantities for biophysical analysis. These two proteins adopt the same three-dimensional structure despite having only 34% sequence identity (30 of 86 residues). Therefore, the HPr systems provide a unique opportunity to test the hierarchical model in two different proteins and to compare the """"""""context-dependence"""""""" of structure formation. Peptides representing the helical portions of these proteins exhibit substantial helix formation as isolated peptides in water. Solvent-exposed, helix-stabilizing interactions will be studied in the two related proteins and compared with results from the isolated peptides to arrive at a complete characterization of the energetics of protein and peptide stability. Other fragments of the HPr proteins will be used to address the role of secondary structure formation in protein stability and to quantify the energetics of the interactions between specific elements of secondary structure. The role of many of the 30 identical residues between the two proteins in defining the HPr fold will be determined through directed mutagenesis studies. The long-term goal is to define the contribution of each amino acid residue to the stability and structure of HPr. The structural ramifications of the mutations, in both the intact proteins and peptide fragments, will be determine by NMR spectroscopy in combination with hydrogen exchange to correlate the structural effects of mutation with the local and global conformational stability. To date, no single system has provided the opportunity to compare the results from structure formation in isolated peptides with intact proteins and to determine the effects of identical mutations in two related protein systems.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Texas A&M University
Schools of Medicine
College Station
United States
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Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R (2014) Forces stabilizing proteins. FEBS Lett 588:2177-84
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