Side chain burial and hydrophobic core packing in protein folding transition states

Abstract

A critical step in the folding pathway of globular proteins is the formation of a tightly packed hydrophobic core. Several mutational studies have addressed the question of whether tight packing interactions are present during the rate-limiting step of folding. In some of these investigations, substituted side chains have been assumed to form native-like interactions in the transition state when the folding rates of mutant proteins correlate with their native-state stabilities. Alternatively, it has been argued that side chains participate in nonspecific hydrophobic collapse when the folding rates of mutant proteins correlate with side-chain hydrophobicity. In a reanalysis of published data, we have found that folding rates often correlate similarly well, or poorly, with both native-state stability and side-chain hydrophobicity, and it is therefore not possible to select an appropriate transition state model based on these one-parameter correlations. We show that this ambiguity can be resolved using a two-parameter model in which side chain burial and the formation of all other native-like interactions can occur asynchronously. Notably, the model agrees well with experimental data, even for positions where the one-parameter correlations are poor. We find that many side chains experience a previously unrecognized type of transition state environment in which specific, native-like interactions are formed, but hydrophobic burial dominates. Implications of these results to the design and analysis of protein folding studies are discussed.

Figure 1.

Experimentally determined changes in transition-state free energies, G_T ^exp, plotted as a function of changes in native-state stability, G_N (A,D), side-chain hydrophobicity, G_H (B,E), and back-calculated values computed using Equation 11, G_T^calc (C,F) for mutations of Ile28 (A,B,C) and Ala39 (D,E,F) in the Fyn SH3 domain. Equations for the lines in A, B, D, and E were obtained by one-parameter linear regression through the origin. Lines in C and F have a slope of 1 and a y-intercept of 0. Error bars corresponding to experimental uncertainties are smaller than the symbols shown.

Figure 2.

Structures of (A) the N-terminal domain of ribosomal protein L9 (NTL9: PDB accession code 1DIV) (Hoffman et al. 1994) and (B) the SH3 domain from the Fyn tyrosine kinase (FYN: PDB accession code 1SHF) (Noble et al. 1993) generated using the MOLMOL software package (Koradi et al. 1996). Side chains participating in specific native-like interactions in the transition state (χ_N > 0) are colored orange. Side chains participating purely in nonspecific hydrophobic collapse (χ_H > 0, χ_N ≈ 0) are colored blue.