Phi-value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy

Abstract

Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion Phi-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify Phi values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. Phi-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the beta(3)-beta(4)-hairpin already is formed in the first transition state, whereas strand beta(1), which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts.

Fig. 1.

Schematic representation of the secondary structure of a homology model of the A39V/N53P/V55L G. gallus Fyn SH3 domain, featuring the characteristic SH3 domain β-sandwich fold formed by the terminal (strands β₁ from Leu-3 to Ala-6 and β₅ from Leu-55 to Pro-57) and the approximately orthogonal central β-sheets (strands β₂ from Asp-25 to Asn-30, β₃ from Trp-36 to Ser-41, and β₄ from Thr-47 to Ile-50), along with a helical turn with 3₁₀ geometry from Pro-51 to Tyr-54. The residues Leu-3, Glu-5, Phe-20, Arg-40, and Thr-47 mutated in this study are shown in ball-and-stick representation. The homology model was created as described previously (7).

Fig. 2.

Changes in ΔG used to calculate Φ_A (Top), along with changes in free energy upon point mutation, ΔΔG, along the folding pathway for several mutants of the A39V/N53P/V55L Fyn SH3 domain based on the parameters of SI Table 3. Values of G are referenced with respect to the unfolded state U that is arbitrarily assigned a value of 0. TS(UI) and TS(IF) denote the rate-limiting transition states between states U, I and I, F respectively. Inset for the A39V/T47S/N53P/V55L mutant shows the pair of ΔG profiles (pseudo-wt and mutant) from which ΔΔG values are obtained, with the free energies of U states both assigned arbitrarily to 0, which is strictly true only if ΔG_U = 0.

Fig. 3.

Comparison of secondary ¹⁵N chemical shifts (ϖ_X − ϖ_RC; ref. , see text) of A39V/R40T/N53P/V55L Fyn SH3 and A39V/N53P/V55L Fyn SH3 for the folded (Top), intermediate (Middle), and unfolded (Bottom) states. Residues in the immediate vicinity of the mutated side chain in the native structure (Fig. 1) are indicated by asterisks.