Comparison of protein backbone entropy and beta-sheet stability: NMR-derived dynamics of protein G B1 domain mutants.

The stability of folded proteins is influenced by the intrinsic propensities of the amino acids to adopt particular secondary structural conformations. Although the statistically observed and experimentally measured propensities correlate well for both R-helices and â-sheets, the physicochemical basis of these propensities remains a topic of discussion.1-3 A correlation between â-sheet propensities and the intrinsic amide-solvent hydrogen exchange rates of the amino acids4 led to the suggestion that higher â-sheet propensity results from the obstruction of solvent-backbone hydrogen bonds in the unfolded states of proteins, thus enthalpically stabilizing the folded relative to the unfolded state. Alternatively, â-sheet propensities can be explained by the influence of local steric interactions on the number of conformations accessible to a given residue in a â-sheet versus a random coil.5 This suggests a strong entropic component to the observed energetic differences in â-sheet formation. In this communication, we report experimental estimates of backbone entropy for three â-sheet mutants of a small (56 amino acid) protein domain, the B1 domain of Streptococcal protein G.6 The B1 domain (Tm ) 89 °C), which consists of a four-stranded â-sheet packed against a single R-helix, has been used previously as a model for determination of intrinsic â-sheet propensities by measurement of the stability of the folded domain after substitution of a surface position in the â-sheet (residue 53) with each of the 20 natural amino acids;2,3 background mutations were also introduced to minimize cross-strand interactions. In the present study we have used the A53, M53, and T53 mutants described by Smith et al.2, chosen for their wide range of stabilities (∆∆Gfolding relative to A53 at 30 °C) of 0, -4.2, and -8.9 kJ.mol-1, respectively. We estimated the backbone entropy of each mutant using an established relationship between entropy and NMR-derived7 order parameters for backbone NH groups in proteins.8,9 The order parameter (S2), describing the amplitude of angularly restricted internal motion, is obtained by fitting NMR relaxation data to the Lipari-Szabo dynamics formalism.10 For the diffusion-in-a-cone model of NH vector motions, the conformational entropy of an isolated NH group is: