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

2021 ◽  
Author(s):  
Sophia Gruber ◽  
Achim Loef ◽  
Adina Hausch ◽  
Res Joehr ◽  
Tobias Obser ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Critical Role ◽  
Coagulation Factor ◽  
Magnetic Tweezers ◽  
The Stability ◽  
And Function ◽  
Von Willebrand

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 D'D3 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 D'D3 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 D'D3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

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

2018 ◽  
Author(s):  
Achim Löf ◽  
Philipp U Walker ◽  
Steffen M Sedlak ◽  
Tobias Obser ◽  
Maria A Brehm ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Protein Domain ◽  
High Yield ◽  
Primary Hemostasis ◽  
Wide Range ◽  
And Function ◽  
Von Willebrand

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.

Von Willebrand disease and Weibel-Palade bodies

2010 ◽  
Vol 30 (03) ◽  
pp. 150-155 ◽  
Author(s):  
J. W. Wang ◽  
J. Eikenboom
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Inflammatory Factors ◽  
Limited Data ◽  
Storage Organelle ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) is a pivotal haemostatic protein mediating platelet adhesion to injured endothelium and carrying coagulation factor VIII (FVIII) in the circulation to protect it from premature clearance. Apart from the roles in haemostasis, VWF drives the formation of the endothelial cell specific Weibel-Palade bodies (WPBs), which serve as a regulated storage of VWF and other thrombotic and inflammatory factors. Defects in VWF could lead to the bleeding disorder von Willebrand disease (VWD).Extensive studies have shown that several mutations identified in VWD patients cause an intracellular retention of VWF. However, the effects of such mutations on the formation and function of its storage organelle are largely unknown. This review gives an overview on the role of VWF in WPB biogenesis and summarizes the limited data on the WPBs formed by VWD-causing mutant VWF.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

scholarly journals Energetic dependencies dictate folding mechanism in a complex protein

2019 ◽  
Vol 116 (51) ◽  
pp. 25641-25648 ◽  
Author(s):  
Kaixian Liu ◽  
Xiuqi Chen ◽  
Christian M. Kaiser
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Large Scale ◽  
Elongation Factor ◽  
Multidomain Protein ◽  
Domain Interactions ◽  
The Stability ◽  
Domain Iii ◽  
And Function ◽  
Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

scholarly journals von Willebrand factor propeptide: biology and clinical utility

Blood ◽  
2015 ◽  
Vol 126 (15) ◽  
pp. 1753-1761 ◽  
Author(s):  
Sandra L. Haberichter
Keyword(s):  
Von Willebrand Factor ◽  
Clinical Utility ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
True Type ◽  
And Function ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Von Willebrand Factor Propeptide

Abstract von Willebrand factor (VWF) is a large multimeric glycoprotein that mediates the attachment of platelets to damaged endothelium and also serves as the carrier protein for coagulation factor VIII (FVIII), protecting it from proteolytic degradation. Quantitative or qualitative defects in VWF result in von Willebrand disease (VWD), a common inherited bleeding disorder. VWF is synthesized with a very large propeptide (VWFpp) that is critical for intracellular processing of VWF. VWFpp actively participates in the process of VWF multimerization and is essential for trafficking of VWF to the regulated storage pathway. Mutations identified within VWFpp in VWD patients are associated with altered VWF structure and function. The assay of plasma VWFpp has clinical utility in assessing acute and chronic vascular perturbation associated with diseases such as thrombotic thrombocytopenic purpura, sepsis, and diabetes among others. VWFpp assay also has clear utility in the diagnosis of VWD subtypes, particularly in discriminating true type 3 subjects from type 1C (reduced plasma survival of VWF), which is clinically important and has implications for therapeutic treatment.

scholarly journals Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses

2021 ◽  
Author(s):  
Claire Gething ◽  
Joshua Ferrar ◽  
Bishal Misra ◽  
Giovanni Howells ◽  
Ucheor B. Choi
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Synaptic Vesicle ◽  
Conformational Change ◽  
Single Molecule ◽  
Conformational Changes ◽  
Snare Complex ◽  
Complex Assembly ◽  
Ribbon Synapses ◽  
Snare Complex Formation

AbstractNeurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

scholarly journals Probing the stability of the SpCas9-DNA complex after cleavage

2021 ◽  
Author(s):  
Pierre Aldag ◽  
Fabian Welzel ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Marius Rutkauskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Slowing Down ◽  
Target Strand ◽  
Dna Strands ◽  
Dna Strand ◽  
The Stability ◽  
The Individual ◽  
Dna Complex

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

Nanoscale Analysis of the Effect of Pathogenic Mutations on Polycystin-1

2010 ◽  
Author(s):  
Liang Ma ◽  
Meixiang Xu ◽  
Andres F. Oberhauser
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Structure And Function ◽  
Eukaryotic Cells ◽  
Mechanical Forces ◽  
Force Microscopy ◽  
Physiological Conditions ◽  
Atomic Force ◽  
And Function

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells is the result of cycles of mechano-sensing, mechano-transduction, and mechano-response. Recently developed single-molecule atomic force microscopy (AFM) techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins, such as titin (an elastic mechano-sensing protein found in muscle) and polycystin-1 (PC1, a mechanosensor found in the kidney).

scholarly journals Dynamics of TRF1 organizing a single human telomere

2020 ◽  
Author(s):  
Xu Li ◽  
Meijie Wang ◽  
Wei Zheng ◽  
Wei Huang ◽  
Zeyu Wang ◽  
...  
Keyword(s):  
Telomere Length ◽  
Single Molecule ◽  
Critical Role ◽  
Magnetic Tweezers ◽  
Critical Force ◽  
Binding Energies ◽  
Future Research ◽  
Telomeric Dna ◽  
Efficient Replication ◽  
Human Telomere

Abstract Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 kBT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.

scholarly journals Probing protein - DNA interaction by single molecule and structural analysis

2014 ◽  
Vol 70 (a1) ◽  
pp. C198-C198
Author(s):  
Jianshi Jin ◽  
Tengfei Lian ◽  
Chan Gu ◽  
Yiqin Gao ◽  
Yujie Sun ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Fundamental Property ◽  
Dna Interaction ◽  
Allosteric Effect ◽  
Dna Protein Interaction ◽  
Fine Tune

In proteins, conformational change impacting their function has been well investigated in the past decades, and was named `allosteric effect'. However, in DNA-protein interaction, the concept of DNA conformational change caused by DNA-protein binding will affect another nearby DNA-binding protein has not been well investigated and understood. Combined with structural biology and Single Molecule Assays, we can now probe and study allosteric propagation through DNA which exists as a fundamental property in DNA-protein interaction, and this allosteric effect through DNA can fine tune gene expression. Therefore, DNA conformational changes should be seriously considered and analyzed for DNA –protein interactions in general.

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