CHARACTERIZATION OF PROTEIN FOLDING INTERMEDIATES (HELIX, KINETICS, NMR, HYDROGEN EXCHANGE)

The three-dimensional structure of a protein is encoded in its linear sequence of amino acids. Studies of protein folding are aimed at understanding the nature of this code which translates one-dimensional information to three-dimensions. It is now well-established that protein folding intermediates exist and can be populated significantly under some conditions.; Here, a method to characterize kinetic folding intermediates is described. The method takes advantage of the decrease in exchange rates between amide protons (i.e., peptide backbone NH) and solvent water protons, when the amide proton is involved in structure. Amide proton exchange rates are monitored using isotopically labeled water, D(,2)O or ('3)H -H(,2)O. An advantage of using amide proton exchange to monitor folding is that the location of exchanged protons, and the extent of exchange for a given proton, can ultimately be determined using nuclear magnetic resonance spectroscopy (NMR), after folding has gone to completion.; The feasibility of using amide proton exchange to pulse-label proteins during folding has been demonstrated using ('3)H -H(,2)O. The results with ribonuclease A (RNase A) support a framework model for folding, in which the secondary structure of a protein is formed before tertiary structure changes are complete. Extension of these studies using NMR should permit characterization of early secondary structure folding frameworks.; The relationship between amino acid sequence and secondary structure has been studied in short fragments of RNase A. Both C-peptide (residues 1-13) and S-peptide (residues 1-20) of RNase A show partial (alpha)-helix formation in aqueous solutions at temperatures near 0(DEGREES)C. Helix formation is strongly dependent on pH in both peptides; the pH dependence has been investigated using chemically synthesized variants of C-peptide that contain amino acid substitutions.; The NMR chemical shifts of various sidechain resonances in S-peptide have been monitored as the helix is unfolded by denaturants or an increase in temperature. The results suggest that only certain residues in S-peptide participate in helix formation, and that a functional helix-termination signal exists in the isolated peptide.