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Single-molecule mechanics and regulatory conformational transitions of the force-sensing protein von Willebrand factor
Single-molecule mechanics and regulatory conformational transitions of the force-sensing protein von Willebrand factor
The formation of hemostatic plugs at sites of vascular injury represents a first essential step in the blood coagulation cascade. This process crucially relies on the large, linear multimeric glycoprotein von Willebrand factor (VWF) and its ability to stably bind and recruit platelets to the damaged vessel wall even under conditions of high shear stress. Remarkably, VWF’s hemostatic activity is regulated by force. Forces on VWF multimers in the bloodstream result from the interplay of their immense lengths (up to ≈ 15 µm) with the hydrodynamic flow they encounter. While being inactive under normal blood flow conditions, VWF is activated for its hemostatic function by increased hydrodynamic forces that result from changes in the blood flow profile in the wake of vascular injury, especially due to an elevated elongational flow component. This force-regulation of VWF’s hemostatic activity is not only highly intriguing from a biophysical perspective, but also of eminent physiological importance. On the one hand, it prevents undesired activity of VWF in intact vessels that could lead to thromboembolic complications. On the other hand, it provides a mechanism to facilitate effcient VWF-mediated platelet aggregation exactly where needed. Prerequisite for activation of a VWF multimer is the force-induced, abrupt transition from a rather compact, overall globular conformation to an elongated, string-like conformation. Importantly, VWF’s elongation behavior is governed by several specific intramolecular interactions and force-induced conformational transitions within VWF’s dimeric subunits. By regulating the effective multimer length, these intramolecular interactions also govern VWF’s initial force sensitivity, as hydrodynamic forces strongly scale with dimension. However, despite their central role in the mechano-regulation of VWF’s hemostatic function, these intramolecular interactions and further regulatory force-induced conformational transitions are for the most part not well understood and characterized. In the framework of this thesis, in order to dissect regulatory conformational transitions governing VWF’s hemostatic activity, the mechanical response and the conformational ensemble of VWF dimers –the smallest repeating subunits of multimers– were investigated at the single-molecule level. Using a combination of atomic force microscopy (AFM) imaging and AFM-based single-molecule force measurements, it was shown that even minor pH changes from the physiologic pH of 7.4, especially acidification, result in a markedly decreased mechanical resistance of VWF’s dimeric subunits. This effect could be traced back to destabilization of a specific, strong intermonomer interaction mediated by VWF’s D4 domains. This pH dependence might represent a mechanism to promote activation of VWF in response to local pH changes, which may occur at sites of vascular injury. In addition, further pH-dependent, but mechanically very weak interactions in the C-terminal stem region of VWF dimers could be inferred from the imaging results. To enable direct investigation of interactions in VWF that dissociate at very low, but physiologically highly relevant forces down to < 1 pN, a novel approach for single-molecule protein force spectroscopy based on magnetic tweezers (MT) was developed. This approach, which enables highly parallel and stable measurements at constant forces, was validated using the well-characterized protein domain ddFLN4 as a model system. In this context, also the lifetime of single biotin–streptavidin bonds was investigated and, by measurements with streptavidin variants of different valencies, it was shown that the bond lifetime strongly depends on the pulling geometry. Applying the MT assay to dimeric VWF constructs, several force-induced conformational transitions in VWF could be characterized. For instance, the impact of calcium binding on the kinetics of unfolding and refolding of the VWF A2 domain, a process relevant both for VWF’s activation and down-regulation, was elucidated. Furthermore, mechanically very weak interactions in the C-terminal stem region of VWF dimers, which had previously only been inferred indirectly, were observed directly at a force of ≈ 1 pN. These interactions can be expected to have important physiological implications, as their dissociation likely represents the first specific step of force-induced elongation of VWF. Moreover, a previously unknown transition within VWF’s N-terminal D’D3 assembly was discovered that likely plays a regulatory role in VWF’s biosynthesis. Finally, single-molecule AFM imaging was introduced as a tool to determine the multimer size distribution of VWF, which, due to the positive relation between multimer length and hydrodynamic force, is highly important for VWF’s overall activity. This approach confirmed the previously described exponential size distribution of VWF and, in particular, yielded insights into clinically relevant multimerization defects that could not be gained by established methods of multimer analysis. Taken together, the findings presented in this thesis help to gain a deeper understanding of the complex interplay of interactions and conformational transitions underlying the force-regulation of VWF’s hemostatic function.
Biophysics, Single-molecule force spectroscopy, Magnetic tweezers, Atomic force microscope, Scanning probe microscopy
Löf, Achim
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Löf, Achim (2019): Single-molecule mechanics and regulatory conformational transitions of the force-sensing protein von Willebrand factor. Dissertation, LMU München: Faculty of Physics
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Abstract

The formation of hemostatic plugs at sites of vascular injury represents a first essential step in the blood coagulation cascade. This process crucially relies on the large, linear multimeric glycoprotein von Willebrand factor (VWF) and its ability to stably bind and recruit platelets to the damaged vessel wall even under conditions of high shear stress. Remarkably, VWF’s hemostatic activity is regulated by force. Forces on VWF multimers in the bloodstream result from the interplay of their immense lengths (up to ≈ 15 µm) with the hydrodynamic flow they encounter. While being inactive under normal blood flow conditions, VWF is activated for its hemostatic function by increased hydrodynamic forces that result from changes in the blood flow profile in the wake of vascular injury, especially due to an elevated elongational flow component. This force-regulation of VWF’s hemostatic activity is not only highly intriguing from a biophysical perspective, but also of eminent physiological importance. On the one hand, it prevents undesired activity of VWF in intact vessels that could lead to thromboembolic complications. On the other hand, it provides a mechanism to facilitate effcient VWF-mediated platelet aggregation exactly where needed. Prerequisite for activation of a VWF multimer is the force-induced, abrupt transition from a rather compact, overall globular conformation to an elongated, string-like conformation. Importantly, VWF’s elongation behavior is governed by several specific intramolecular interactions and force-induced conformational transitions within VWF’s dimeric subunits. By regulating the effective multimer length, these intramolecular interactions also govern VWF’s initial force sensitivity, as hydrodynamic forces strongly scale with dimension. However, despite their central role in the mechano-regulation of VWF’s hemostatic function, these intramolecular interactions and further regulatory force-induced conformational transitions are for the most part not well understood and characterized. In the framework of this thesis, in order to dissect regulatory conformational transitions governing VWF’s hemostatic activity, the mechanical response and the conformational ensemble of VWF dimers –the smallest repeating subunits of multimers– were investigated at the single-molecule level. Using a combination of atomic force microscopy (AFM) imaging and AFM-based single-molecule force measurements, it was shown that even minor pH changes from the physiologic pH of 7.4, especially acidification, result in a markedly decreased mechanical resistance of VWF’s dimeric subunits. This effect could be traced back to destabilization of a specific, strong intermonomer interaction mediated by VWF’s D4 domains. This pH dependence might represent a mechanism to promote activation of VWF in response to local pH changes, which may occur at sites of vascular injury. In addition, further pH-dependent, but mechanically very weak interactions in the C-terminal stem region of VWF dimers could be inferred from the imaging results. To enable direct investigation of interactions in VWF that dissociate at very low, but physiologically highly relevant forces down to < 1 pN, a novel approach for single-molecule protein force spectroscopy based on magnetic tweezers (MT) was developed. This approach, which enables highly parallel and stable measurements at constant forces, was validated using the well-characterized protein domain ddFLN4 as a model system. In this context, also the lifetime of single biotin–streptavidin bonds was investigated and, by measurements with streptavidin variants of different valencies, it was shown that the bond lifetime strongly depends on the pulling geometry. Applying the MT assay to dimeric VWF constructs, several force-induced conformational transitions in VWF could be characterized. For instance, the impact of calcium binding on the kinetics of unfolding and refolding of the VWF A2 domain, a process relevant both for VWF’s activation and down-regulation, was elucidated. Furthermore, mechanically very weak interactions in the C-terminal stem region of VWF dimers, which had previously only been inferred indirectly, were observed directly at a force of ≈ 1 pN. These interactions can be expected to have important physiological implications, as their dissociation likely represents the first specific step of force-induced elongation of VWF. Moreover, a previously unknown transition within VWF’s N-terminal D’D3 assembly was discovered that likely plays a regulatory role in VWF’s biosynthesis. Finally, single-molecule AFM imaging was introduced as a tool to determine the multimer size distribution of VWF, which, due to the positive relation between multimer length and hydrodynamic force, is highly important for VWF’s overall activity. This approach confirmed the previously described exponential size distribution of VWF and, in particular, yielded insights into clinically relevant multimerization defects that could not be gained by established methods of multimer analysis. Taken together, the findings presented in this thesis help to gain a deeper understanding of the complex interplay of interactions and conformational transitions underlying the force-regulation of VWF’s hemostatic function.