To establish a framework for extrapolating the helix-forming properties of peptides from TFE/H2O mixtures (TFE = 2,2, 2-trifluoroethanol) back to water, the thermal unfolding curves have been measured by circular dichroism for four repeating-sequence peptides, with chain lengths from 7 to 22 residues. The unfolding curves were measured between 0 and 50 volume percent TFE and were fitted to the modified Lifson-Roig theory. A single set of helix-coil parameters fits the results for the four peptides at each TFE concentration; only two of the basic helix-coil parameters, , the mean helix propagation parameter of residues in the sequence repeat, and DeltaH, the enthalpy change per residue on unfolding the helix, are allowed to vary with TFE molarity. The success in fitting these curves over a wide range of experimental variables shows that helix formation is basically the same in TFE/H2O mixtures as in water. Moreover, a simple model based on a linear dependence of ln and DeltaH on TFE molarity can be used to extrapolate the results from 25% TFE (approximately 4 M) back to water. The results also give curves of helix formation induced by TFE at constant temperature, and the properties of these helix induction curves explain some of the puzzling results shown by other peptides in the literature. The average helix propensity increases regularly from 0 to 25% TFE but levels off at higher TFE concentrations, which explains why the extent of helix formation levels off in this range. The change in the apparent cooperativity of thermal unfolding curves in concentrated TFE solutions results from the decrease of the enthalpy change for helix unfolding at higher TFE concentrations. The rapid decrease in the plateau values of apparent helix content with increasing temperature results mainly from the strong temperature dependence of the ellipticity of the complete helix. To determine whether the helix-stabilizing effect of TFE arises from strengthening the hydrogen bonds in the helix backbone, the strength of the hydrogen bond in a model compound, salicylic acid, has been measured in TFE/H2O mixtures from the pKa difference between salicylic acid and a similar compound which cannot form the hydrogen bond. The curve of hydrogen bond strength versus increasing TFE concentration matches both in shape and magnitude the increase in average helix propensity in TFE/H2O mixtures.

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (؊1.0 kcal͞mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as Ϸ؊11.5 kcal͞mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is ؊4.9 kcal͞mol. These numbers give an enthalpy deficit for helix formation of ؊7.6 kcal͞mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and ␤-strand conformations, by using the DELPHI program and PARSE parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the ␤-strand conformation, the ESF value of an interior alanine peptide group is ؊7.9 kcal͞mol, substantially less than that of N-methylacetamide (؊12.2 kcal͞mol), and the helical peptide group is solvated with an ESF of ؊2.5 kcal͞mol. These results reduce the enthalpy deficit to ؊1.5 kcal͞mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.B aker and Hubbard (1) found that water molecules cluster around peptide CAO groups in protein x-ray structures. Ben-Naim (2) and Honig and coworkers (3, 4) used model compound data to argue that interaction of water with peptide CAO groups in protein secondary structures should be an important factor in the energetics of protein folding. If the free energy of the interaction is as small as Ϫ0.5 kcal͞mol, and if half the peptide groups in a 100-residue protein are stripped of water molecules when folding is complete, then breaking the interactions between water and peptide CAO groups should cost 25 kcal. If some of these water-peptide interactions are not broken in forming a molten globule folding intermediate, they would represent a very important source of stabilizing free energy.The alanine peptide helix provides a suitable model system for determining the strength of the interaction between water and the helical peptide group and for examining the effects of side chains on this interaction. Modeling studies indicate that side chains can block the access of water to backbone CAO groups in a helix (5, 6) in a side chain-specific and rotamer-specific (6) manner. Formation of the alanine helix is known to be enthalpydriven with an enthalpy change of Ϫ1.0 kcal͞mol per residue (7,8). A high-resolution x-ray structure of an alanine peptide helix is available (9). We show below that if amides such as N-methylacetamide are used as models for the free peptide group in an unfolded alanine peptide, then the...

We report an enthalpic factor involved in determining helix propensities of nonpolar amino acids. Thermal unfolding curves of the five 13-residue peptides, Ac-KA 4 XA 4 KGY-NH 2 (X ‫؍‬ Ala, Leu, Ile, Val, Gly), have been measured by using CD in water͞trif luoroethanol (TFE) mixtures. The peptide helix contents show that the rank order of helix propensities changes with temperature: although Ala has the highest helix propensity at 0°C in all TFE concentrations, it is lower than Leu, Ile, and Val at 50°C in 20% TFE. This change is attributed to shielding by nonpolar side chains of the interaction between water and polar groups in the helix backbone for the following reasons. (i) Helix content is directly related to helix propensity for these designed peptides because side-chain-side-chain interactions are absent. (ii) The change in rank order with temperature is enthalpic in origin: in water, the apparent enthalpy of helix formation calculated from the thermal unfolding curves varies widely among the five peptides and has the same rank order as the helix propensities at 0°C. The rank order does not result from burial of nonpolar surface area because the calculated heat capacity change (⌬Cp) on helix formation is opposite in sign from the expected ⌬Cp. (iii) A nonpolar side chain can exclude water from interacting with helix polar groups, according to calculations of water-accessible surface area, and the polar interaction between water and peptide polar groups is entirely enthalpic, as shown by amide transfer data.There are basic reasons for believing that a still unknown factor, in addition to side-chain entropy, is important in determining the values of the helix propensities of the nonpolar amino acids. Only nonpolar residues are considered because polar and charged amino acids present a more complex case. The first reason is that, although the rank order is the same in both systems, the relative helix propensities are quantitatively different in alanine-based peptides (1) and in peptide sequences from protein helices (2, 3). The helix propensity of alanine is 35 times greater than that of glycine in alanine-based peptides (1) but only six times greater in an RNase T1 peptide helix (3) or in RNase T1 itself (3), or in a compilation of data for 323 peptides taken from the literature and analyzed by the algorithm AGADIR (2). The second reason is that, although helix propensities of the nonpolar amino acids are highly correlated with the loss of side-chain conformational entropy that occurs upon helix formation (4-6), nonetheless this effect accounts only for one-third of the free energy differences arising from the helix propensities of the nonpolar amino acids (1).Consequently, we undertook a search for an unknown factor that plays a dominant role in determining the values of helix propensities. Our procedure and its rationale are as follows. First, there are good reasons for believing that the context dependence of the helix propensities arises from the immediate neighboring four residues on either side...