Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding

Abstract

Many experimental and theoretical studies have suggested a significant role for nonnative interactions in protein folding pathways, but the energetic contributions of these interactions are not well understood. We have addressed the energetics and the position specificity of nonnative hydrophobic interactions by developing a continuum coarse-grained chain model with a native-centric potential augmented by sequence-dependent hydrophobic interactions. By modeling the effect of different hydrophobicity values at various positions in the Fyn SH3 domain, we predicted energetically significant nonnative interactions that led to acceleration or deceleration of the folding rate depending on whether they were more populated in the transition state or unfolded state. These nonnative contacts were centered on position 53 in the Fyn SH3 domain, which lies in an exposed position in a 3(10)-helix. The energetic importance of the predicted nonnative interactions was confirmed experimentally by folding kinetics studies combined with double mutant thermodynamic cycles. By attaining agreement of theoretical and experimental investigations, this study provides a compelling demonstration that specific nonnative interactions can significantly influence folding energetics. Moreover, we show that a coarse-grained model with a simple consideration of hydrophobicity is sufficient for the accurate prediction of kinetically important nonnative interactions.

Fig. 1.

A ribbon drawing of the WT Fyn SH3 domain (PDB structure 1SHF). The structured region of the folding transition state of the domain as deduced from Φ-value analyses is highlighted in gray. The side chains of N53 (blue) and several other residues (red) investigated in this study are shown.

Fig. 2.

Effects of increasing the hydrophobicity of specific sites on the folding rate of the Fyn SH3 domain. (A) Simulated folding rates k_f in units of reciprocal number of Langevin time steps, as functions of individual hydrophobicity κ_i for 12 amino acid positions. The folding rates here correspond to that of single mutants of WT Fyn SH3 with a hydrophobic residue hφ of variable κ_i substituted for the WT amino acid residue at the sequence positions indicated (e.g., the N53 curve is for an N53hφ mutant of WT with variable κ₅₃). (B) The correlation between folding transition state stability of N53 mutants (mut) calculated by using the experimental rates (Table S1) and side-chain hydrophobicity. The plotted quantities are normalized by that of the alanine (Ala) mutant, namely, ΔΔG_‡→u = −RT ln(k_f^mut/k_f^Ala), and the corresponding change in side-chain hydrophobicity ΔΔG_hφ = ΔG_hφ^mut − ΔG_hφ^Ala, where ΔG_hφ is the hydrophobicity values of Fauchère and Pliška (33) and the meanings of the superscripts are as noted above. ΔG_‡→u corresponds to the height (>0) of the free energy barrier to folding in our notation (17, 30), although only mutational changes of ΔG_‡→u were considered in this study. (C) Simulated folding rates as functions of individual hydrophobicity κ_i for 10 amino acid positions (as indicated) in a hφ53 background with κ₅₃ = 2.0 (e.g., the N53hφ-D16 curve here is for an N53hφ, D16hφ double mutant of WT with a constant κ₅₃ = 2.0 and variable κ₁₆).

Fig. 3.

Simulated contact probability maps of the folding transition state ensemble of N53 mutants of Fyn SH3 domain. (A) Contact probabilities for κ₅₃ = 1.0. (B) Contact probabilities for κ₅₃ = 3.0. (C ) Differences between A and B, i.e., contact probabilities for κ₅₃ = 3.0 minus that for κ₅₃ = 1.0, highlighting amino acid positions interacting more favorably with hφ53 as its hydrophobicity (κ₅₃) increases. In these maps, two residues, ij, are considered to be in contact if r_ij < 8 Å, irrespective of whether the contact is native or nonnative.

Fig. 4.

Thermodynamic cycles to demonstrate nonnative interactions in the folding transition state of N53 mutants. The number along each arrow is the experimentally measured change in the transition-to-unfolded free energy difference, ΔΔG_‡→u, in units of kcal·mol⁻¹, resulting from the mutation indicated by the arrow (see Fig. 2 legend). Each boxed value corresponds to the interaction energy (ΔG_int), which is equal to the ΔΔG_‡→u value for the vertical arrow on the left minus that on the right, with uncertainty estimated by using the method in the text and SI Methods. ΔG_int > 0 or ΔG_int < 0 is indicative, respectively, of stabilization or destabilization of the folding transition state. The double-mutant cycles here probe interactions between L3 and I53 (A), F4 and I53 (B), L3 and N53 (C ), F4 and N53 (D), positions 40 and 53 (E ), and positions 47 and 53 (F ).

Fig. 5.

Populations of specific nonnative hydrophobic interactions along the folding pathway of Fyn SH3 domain. Results shown here are for the hφ53–hφ3 (A) and hφ53–hφ47 (B) interactions, obtained by simulating the behaviors of two double mutants (e.g., N53I-L3I and N53I-T47I) with κ₅₃ = κ₃ = 2.0 (A) and κ₅₃ = κ₄₇ = 2.0 (B), respectively, whereas other κ_i values are kept identical to that of WT. The progress variable Q is the total number of native contacts in a conformation normalized by the corresponding number in the fully folded PDB structure. In the determination of Q during simulations (13, 25), two residues, ij, are in contact if r_ij < 1.2 r_ijⁿ, where r_ijⁿ is their C_α–C_α distance in the PDB structure. (Upper) The probabilities P_53–3(Q) and P_53–47(Q) (red curves, right vertical scales) of forming the 53–3 (A) and 53–47 (B) nonnative contacts, respectively, among conformations of a given Q are superposed with free energy profiles of the corresponding proteins at approximately their transition midpoints (green curves, left vertical scales). Here P(Q) denotes the probability distribution of native contact; hence −ln P(Q) is free energy in units of k_BT, where k_BT is Boltzmann constant times absolute temperature (25). (Lower) Example structures in the conformational ensembles containing the given nonnative interactions; positions for the contacting nonnative pairs are marked. The Q values for these two example conformations are 0.56 (A) and 0.21 (B).