Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.

The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Escherichia coli has been determined using a combination of thermal unfolding and urea denaturation experiments. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy, and heat capacity that accompany the equilibrium folding of HPr over a wide range of temperature and urea concentrations. In moderate concentrations of urea, HPr undergoes both high- and low-temperature unfolding, allowing for a reliable determination of the change in heat capacity for the conformational transition. The data are consistent with the linear free energy relationship commonly employed to analyze protein denaturation data, even over a relatively large temperature and urea concentration range. Furthermore, we find that a temperature-independent delta Cp is adequate to describe HPr stability over the accessible temperature range. Finally, our data allow us to evaluate the energetics of the urea-protein interaction. For HPr, the changes in excess enthalpy and entropy of the denaturant-protein interaction(s) make only minor contributions to the observed delta H and delta S terms, presumably due in some part to the small size of the HPr protein.

The energetics of the interaction between the polar side chains of glutamate or aspartate with lysine and glutamate with histidine have been determined using a model alanine-based peptide helix. An evaluation of the effects of NaCl and pH on the interactions between these acidic and basic residues in several different orientations and spacings in an alpha-helical peptide has been made. For many of the peptides, we find a considerable interaction between the polar side chains. In general, the shorter side chains show stronger interactions, but there are more restrictions on the precise geometry of the interactions as dictated by the spacing and orientation of the polar residues in the alpha-helical peptide. The energetics of the interaction between the fully-charged ion pairs can be diminished by added salt, but the interaction is not completely screened even at 2.5 M NaCl. The strength of the interaction between a charged and neutral side chain is not as sensitive to the ionic strength of the solution, suggesting that solvent-exposed hydrogen bonds are forming. All the interactions between the polar residues employed here stabilize helix formation, suggesting that solvent-exposed ion pairs and hydrogen bonds can contribute to the conformational stability of proteins and peptides.

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