Biology subjects

Obser, T.

Publications and source records attributed to Obser, T..

3 recordsLinked to original sources

bioRxiv2026

Magnetic Tweezers Experiments Reveal Increased Mechanical Sensitivity of the F2561Y Mutant in Von Willebrand Factor

The large glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis. By sensing hydrodynamic forces in the bloodstream, VWF elongates under elevated shear, increasing its adhesiveness to platelets. This mechanosensitive behavior arises from its multidomain architecture, where distinct structural elements respond at different force scales. The stem, formed by interactions between C-domains of adjacent monomers in VWF dimers, opens at relatively low forces (~1 pN), representing an early step in force activation. The F2561Y mutation in the C4 domain is a gain-of-function mutation associated with increased shear sensitivity and myocardial infarction, yet its effect on VWF mechanics in the physiologically relevant low-force regime remains unresolved. Here, we combine single-molecule magnetic tweezers with AFM imaging to investigate how F2561Y alters VWF stem dynamics. Magnetic tweezers measurements reveal that A2 domain unfolding remains unchanged in the mutant, whereas stem opening transitions occur at significantly lower forces compared to wildtype. The midpoint force for stem opening is reduced by approximately one order of magnitude, indicating a pronounced destabilization of the compact stem conformation. AFM imaging independently confirms a higher population of open stem states in the mutant, even without applied force. Our findings demonstrate that F2561Y selectively destabilizes the VWF stem and increases its force sensitivity without perturbing global domain stability, providing a direct molecular explanation for its enhanced shear responsiveness and prothrombotic phenotype. More broadly, our work establishes magnetic tweezers as a powerful approach to resolve low-force conformational equilibria in mechanosensitive proteins and to dissect the mechanical impact of disease-associated mutations. HighlightsO_LIThe F2561Y mutation lowers the force required to open the VWF stem. C_LIO_LIF2561Y mutant VWF dimers exhibit more open conformations compared to wildtype. C_LIO_LIForce sensitivity provides a mechanism for the prothrombic phenotype of F2561Y. C_LI

biophysics↗

bioRxiv2021

A Conformational Transition of Von Willebrand Factor's D'D3 Domain Primes It For Multimerization

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the (trans-)Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the DD3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose two previously buried cysteines that are known to be vital for multimerization. By characterizing the conformational change at varying levels of force, we are able to quantify the kinetics of the transition and the stability of the interface. We find a pronounced destabilization of the interface upon lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the DD3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII (FVIII), providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the DD3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

biophysics↗

bioRxiv2018

Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

biophysics↗