Person: Yang, Darren
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Yang
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Darren
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Yang, Darren
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Publication Flow-induced elongation of von Willebrand factor precedes tension-dependent activation(Nature Publishing Group UK, 2017) Fu, Hongxia; Jiang, Yan; Yang, Darren; Scheiflinger, Friedrich; Wong, Wesley; Springer, TimothyVon Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor–ligand binding.Publication The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo(Oxford University Press, 2015) Danilowicz, Claudia; Yang, Darren; Kelley, Craig; Prévost, Chantal; Prentiss, MaraRecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.Publication Multiplexed single-molecule force spectroscopy using a centrifuge(Nature Publishing Group, 2016) Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, WesleyWe present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.Publication Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure(Oxford University Press, 2015) Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, MaraRecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results.Publication Exploring Biomolecular Interactions Through Single-Molecule Force Spectroscopy and Computational Simulation(2016-04-11) Yang, Darren; Joshi, Neel; Kaxiras, Efthimios; Prentiss, Mara; Wong, Wesley P.Molecular interactions between cellular components such as proteins and nucleic acids govern the fundamental processes of living systems. Technological advancements in the past decade have allowed the characterization of these molecular interactions at the single-molecule level with high temporal and spatial resolution. Simultaneously, progress in computer simulation has enabled theoretical research at the atomistic level, assisting in the interpretation of experimental results. This thesis combines single-molecule force spectroscopy and simulation to explore inter- and intra-molecular interactions. Specifically, we investigate the interaction between RecA and DNA to elucidate the underlying molecular mechanism of the DNA homologous recombination process. We also evaluate the stability of the von Willebrand Factor (vWF) A2 domain to determine the molecular origins of von Willebrand Diseases (vWD). This thesis also describes the development and application of a new single-molecule technique that combines the centrifuge force microscope (CFM) with DNA self-assembled mechanical switches to enable massively parallel repeating force measurements of molecular interactions.Publication Complementary strand relocation may play vital roles in RecA-based homology recognition(Oxford University Press, 2012) Peacock-Villada, Alexandra; Yang, Darren; Danilowicz, Claudia; Feinstein, Efraim; Pollock, Nolan; McShan, Sarah; Coljee, Vincent; Prentiss, MaraRecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.