Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations

Abstract

Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments. Tryptophan-cysteine quenching is introduced as an experimental approach with minimal labeling for characterizing such interactions between proteins due to its sensitivity to nano- to microsecond dynamics on subnanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations for interpretation of the experimental observables and to gain molecular-level insights. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments can differentiate between overall attractive and repulsive interactions between different proteins, and they can discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions.

Figure 1.

Experimental measurements of Trp triplet lifetime in villin variants and SH3 in the presence of protein G. Decays are shown as absorbance vs. time (top) and as the derivatives of the decays vs. log(t) calculated as the slope of a linear fit over a sliding window of 21 time points (bottom). (A) K33W at various concentrations of protein G. (B) Villin headpiece wild-type (black), V10W (red), K33W (blue), R15T+K30E (green) and SH3 (magenta). Curves were averaged from six independent measurements and normalized to 0.88 at 147 ns and to 0 at 369 μs. Error bars represent the standard deviation. Error bars in the derivatives represent the error of the fit.

Figure 2.

Computational modeling of Trp-Cys quenching via 1D potentials. Probability distribution functions are shown on the left (A, C, E) and derivatives of the calculated quenching curves are shown on the right (B, D, F). The reference distribution in black in A-D is based on the contact probability extracted from atomistic simulations for the wild-type villin structure (E). Variations in the potential near the contact minimum are shown in A and C. Variations in long-range attraction (d=−1 in Eq. S1, red) or repulsion (d=1 in Eq. S1, green) are shown in C and D. Trp-Cys contact probability functions from atomistic simulations are shown in E (solid lines) along with fitted 1D potentials (dashed lines). Atomistic probabilities are for minimum villin-protein G Trp-Cys distances. Distances are limited to 25 Å since there is almost always a protein G molecule sufficiently close for minimum distances to be less than 30 Å due to crowding. Derivatives on the left in F were obtained with uniform Monte Carlo sampling, derivatives on the right in F resulted from sampling with diffusion-matched, distance-dependent step sizes (see Supplementary Material). Colors in E and F reflect different probes: villin wild-type (black), villin V10W (red), villin K33W (blue), villin R15T+K30E (green), SH3 (magenta).

Figure 3.

Comparison of survival probability decays between experimental measurements and simulated data. The probability and its derivative against log-time are shown at top and bottom of each panel, respectively. Derivative values were obtained from a linear-fit of probabilities against log-time with a window size of 21. Experimental values shown as solid lines with transparent shades for standard errors were adjusted to match the simulated data at 100–500 ns since the initial decay varies in the simulations and experiments are not sensitive to quenching during the initial 100 ns after excitation. A: Results for wild-type villin obtained using different force fields c36 (orange), c36+water (lime), c36m (black), and c36mw (cyan); B: Results with c36m for wild-type villin (black), V10W (red), K33W (blue), and the R15T+K30E double mutant (green); C: Results with c36m for wild-type villin (black) and SH3 (magenta).

Figure 4.

Residue-wise contacts between protein G and villin variants, projected onto the villin surface (A-D) and as a function of villin residue index (E, F). Surface projections are shown for Trp-Cys quenching contact (left) and for all contact positions (right) for wild-type villin (A), the R15T+K30E mutant (B), the V10W mutant (C), and the K33W mutant (D). The location of the Trp residue is indicated by arrows. Contacts per frame vs. residue index are shown at the time of quenching contact (E) and at any time of contact (F) with different variants colored as in Fig. 1. Shaded areas indicate standard errors. Contacts were defined by residue pairs whose inter-atomic distances were closer than 5 Å.