Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)­materials under load and related chemistries.

Protonation states of ionizable amino acid residues such as histidine depend on pH and define the spatial distribution of the electrostatic potential inside protein molecules. They thus play a key role in many biological processes, such as enzyme catalysis, protein dynamics and inter-and intra-molecular interactions. However, standard force fields used in molecular dynamics (MD) simulations do not allow protonation state changes, which can occur in response to a conformational change in a protein. Therefore, many so called constant pH methods for dynamic protonation have been developed [1-3], in which a proton appears or disappears on titratable amino acid residues during MD simulations. Here, we compare the pKa values obtained via our recent constant pH MD implementation to measured NMR titration curves in order to assess the accuracy of our method. We chose a set of pentapeptides with challenging charge compositions yet are small enough to ensure exhaustive sampling in our MD simulations. Both measured and calculated titration curves and pKa values agree well with each other, differing by less than 0.25 pKa units.

The multimeric plasma glycoprotein von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand Disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet glycoprotein (GP)IIb/IIIa-dependent prothrombotic gain-of-function (GOF) for variant p.Pro2555Arg, located in the C4-domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small angle X-ray scattering, NMR spectroscopy, molecular dynamics simulations on the single C4-domain and dimeric wildtype and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF is not affecting the binding affinity of the C4-domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and show a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but which is not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4-domain as a novel anti-thrombotic drug target.