THE MAGNITUDE OF THE BACKBONE CONFORMATIONAL ENTROPY CHANGE IN PROTEIN-FOLDING

The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analy sis. Each pair of mutants differed only by having alanine or glycine a t a specific solvent-exposed position under conditions in which the di fferences in stability could be attributed to differences in the confo rmational entropy of the unfolded state. The mutants studied were char acterized by different stabilities but had identical heat capacity cha nges of unfolding (Delta C-p), identical solvent-related entropies of unfolding (Delta S-solv), and identical enthalpies of unfolding (Delta H) at equivalent temperatures. Accordingly, the differences in stabil ity between the different mutants could be attributed to differences i n conformational entropy. The computational studies were aimed at gene rating the energy profile of backbone conformations as a function of t he main chain dihedral angles phi and phi, The energy profiles permit a direct calculation of the probability distribution of different conf ormers and therefore of the conformational entropy of the backbone. Th e experimental results presented in this paper indicate that the prese nce of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 +/- 0.2 cal/K . mol with respect to th at of glycine, consistent with a 3.4-fold reduction in the number of a llowed conformations in the alanine-containing peptides. Similar resul ts were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side c hain had only a small effect as long as the side chains were unbranche d at position beta. A further reduction with respect to Ala of only 0. 61 and 0.81 cal/K . mol in the backbone entropy was obtained for leuci ne and lysine, respectively. beta-branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K . mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an alpha-helix is 6.51 cal/K . mol for glycine. These and previous res ults have allowed a complete estimation of the conformational entropy changes associated with protein folding. (C) 1996 Wiley-Liss, Inc.