LAUSR

Protein stability can be quantified, in experiments or simulations, by monitoring changes in free energy induced by single amino-acid mutations relative to changes imposed by the same mutation in a reference unfolded peptide. We here assessed the destabilization by disease mutations of a protein, for which both the folded and unfolded states play key functional roles in hemostasis. Force-induced unfolding of the giant von Willebrand Factor (VWF) multimer-protein exposes a cleavage site for enzymatic proteolysis, a critical down-regulatory mechanism to prevent the formation of large thrombus aggregates. Several naturally occurring mutations modify this process, inducing distinct types of bleeding disorders, by unknown mechanisms. We present the first quantitative description of the dramatic destabilization of VWF caused by a one of such mutations, which strongly accelerates VWF cleavage. Molecular dynamics simulations and free energy calculations revealed this mutation to induce structural, dynamic, and mechanical perturbations in the VWF-A2 domain, thereby destabilizing this domain by ∼10 kJ/mol promoting its unfolding. In close agreement, fluorescence correlation spectroscopy (FCS) revealed a 20-fold increase in the cleavage rate for this mutant, compared to the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites at the same time. Taken together, the enhanced cleavage activity can be readily explained by an increased availability of the cleavage site through A2-domain-fold thermodynamic destabilization. Our study therefore puts forward the combination of free energy calculations and FCS, as a powerful way of examining protein stability in a clinically relevant context. Reference: C. Aponte-Santamaria, et al. Biophysical Journal. In revision.