O to bR transition in bacteriorhodopsin occurs through a proton hole mechanism.

Extensive classical and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations are used to establish the structural features of the O state in bacteriorhodopsin (bR) and its conversion back to the bR ground state. The computed free energy surface is consistent with available experimental data for the kinetics and thermodynamics of the O to bR transition. The simulation results highlight the importance of the proton release group (PRG, consisting of Glu194/204) and the conserved arginine 82 in modulating the hydration level of the protein cavity. In particular, in the O state, deprotonation of the PRG and downward rotation of Arg82 lead to elevated hydration level and a continuous water network that connects the PRG to the protonated Asp85. Proton exchange through this water network is shown by ∼0.1-µs semiempirical QM/MM free energy simulations to occur through the generation and propagation of a proton hole, which is relayed by Asp212 and stabilized by Arg82. This mechanism provides an explanation for the observation that the D85S mutant of bacteriorhodopsin pumps chloride ions. The electrostatics-hydration coupling mechanism and the involvement of all titration states of water are likely applicable to many biomolecules involved in bioenergetic transduction.

Mechanism of proton-coupled electron transfer described with QM/MM implementation of coupled-perturbed density-functional tight-binding.

Coupled-perturbed equations for degenerate orbitals were implemented for third order density-functional tight binding, which allowed the use of Mulliken charges as reaction coordinates. The method was applied to proton-coupled electron transfer (PCET) reactions in a model system and thoroughly tested for QM and QM/MM setups (i.e., coupled quantum and molecular mechanics). The performed enhanced sampling simulations were stable, and the obtained potentials of the mean force were able to address the thermodynamic and kinetic features of the reactions by showing the expected topography and energy barriers. Hence, this method has the potential to distinguish between concerted and sequential mechanisms and could next be applied to proton-coupled electron transfer reactions in more complex systems like proteins.

Reduction pathway of glutaredoxin 1 investigated with QM/MM molecular dynamics using a neural network correction.

Glutaredoxins are small enzymes that catalyze the oxidation and reduction of protein disulfide bonds by the thiol-disulfide exchange mechanism. They have either one or two cysteines in their active site, resulting in different catalytic reaction cycles that have been investigated in many experimental studies. However, the exact mechanisms are not yet fully known, and to our knowledge, no theoretical studies have been performed to elucidate the underlying mechanism. In this study, we investigated a proposed mechanism for the reduction of the disulfide bond in the protein HMA4n by a mutated monothiol Homo sapiens glutaredoxin and the co-substrate glutathione. The catalytic cycle involves three successive thiol-disulfide exchanges that occur between the molecules. To estimate the regioselectivity of the different attacks, classical molecular dynamics simulations were performed and the trajectories analyzed regarding the sulfur-sulfur distances and the attack angles between the sulfurs. The free energy profile of each reaction was obtained with hybrid quantum mechanical/molecular mechanical metadynamics simulations. Since this required extensive phase space sampling, the semi-empirical density functional tight-binding method was used to describe the reactive cysteines. For an accurate description, we used specific reaction parameters fitted to B3LYP energies of the thiol-disulfide exchange and a machine learned energy correction that was trained on coupled-cluster single double perturbative triple [CCSD(T)] energies of thiol-disulfide exchanges. Our calculations show the same regiospecificity as observed in the experiment, and the obtained barrier heights are about 12 and 20 kcal/mol for the different reaction steps, which confirms the proposed pathway.

Electrostatic interactions contribute to the control of intramolecular thiol-disulfide isomerization in a protein.

The roles of structural factors and of electrostatic interactions with the environment on the outcome of thiol-disulfide exchange reactions were investigated in a mutated immunoglobulin domain (I27*) under mechanical stress. An extensive ensemble of molecular dynamics trajectories was generated by means of QM/MM simulations for a total sampling of 5.7 µs. A significant number of thiol-disulfide exchanges were observed, and the Cys32 thiolate preferred to attack Cys55 over Cys24, in agreement with previous experimental and computational studies. The structural features as well as electronic structures of the thiol-disulfide system along the reaction were analyzed, as were the electrostatic interactions with the environment. The previous findings of better accessibility of Cys55 were confirmed. Additionally, the reaction was found to be directed by the electrostatic interactions of the involved sulfur atoms with the molecular environment. The relationships of atomic charges, which stem from the electrostatic interactions, lead to the kinetic preference of the attack on Cys55. Further, QM/MM metadynamics simulations of thiol-disulfide exchange in a small model system with varied artificial external electric potentials revealed changes in reaction kinetics of the same magnitude as in I27*. Therefore, the electrostatic interactions are confirmed to play a role in the regioselectivity of the thiol-disulfide exchange reactions in the protein.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding.

BACKGROUND: The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. OBJECTIVE: To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF's platelet binding function. METHODS: We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. RESULTS: We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. CONCLUSIONS: Our data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

Accurate Free Energies for Complex Condensed-Phase Reactions Using an Artificial Neural Network Corrected DFTB/MM Methodology.

Semiempirical methods like density functional tight-binding (DFTB) allow extensive phase space sampling, making it possible to generate free energy surfaces of complex reactions in condensed-phase environments. Such a high efficiency often comes at the cost of reduced accuracy, which may be improved by developing a specific reaction parametrization (SRP) for the particular molecular system. Thiol-disulfide exchange is a nucleophilic substitution reaction that occurs in a large class of proteins. Its proper description requires a high-level ab initio method, while DFT-GAA and hybrid functionals were shown to be inadequate, and so is DFTB due to its DFT-GGA descent. We develop an SRP for thiol-disulfide exchange based on an artificial neural network (ANN) implementation in the DFTB+ software and compare its performance to that of a standard SRP approach applied to DFTB. As an application, we use both new DFTB-SRP as components of a QM/MM scheme to investigate thiol-disulfide exchange in two molecular complexes: a solvated model system and a blood protein. Demonstrating the strengths of the methodology, highly accurate free energy surfaces are generated at a low cost, as the augmentation of DFTB with an ANN only adds a small computational overhead.