Browsing by Author "Kiang, Ching-Hwa"
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Item Advanced Computational Methods for Macromolecular Modeling and Structure Determination(2013-12-05) Zhang, Chong; Ma, Jianpeng; Nordlander, Peter J.; Kiang, Ching-Hwa; Raphael, Robert M.As volume and complexity of macromolecules increase, theories and algorithms that deal with structure determination at low X-ray resolution are of particular importance. With limited diffraction data in hand, experimentalists rely on advanced computational tools to extract and utilize useful information, seeking to determinate a three dimensional model that best fits the experiment data. Success of further studies on the property and function of a specific molecule - the key to practical applications - is therefore heavily dependent on the validity and accuracy of the solved structure. In this thesis I propose Deformable Complex Network (DCN) and introduce Normal Mode Analysis (NMA), which are designed to model the average coordinates of atoms and associated fluctuations, respectively. Their applications on structure determination target two major branches ? the positional refinement and temperature factor refinement. I demonstrate their remarkable performance in structure improvements based on several criteria, such as the free R value, overfitting effect and Ramachandran Statistics, with tests carried out across a broad range of real systems for generality and consistency.Item Analyzing Single-Molecule Manipulation Experiments(2008-10) Calderon, Christopher P.; Harris, Nolan C.; Kiang, Ching-Hwa; Cox, Dennis D.Single-molecule manipulation studies can provide quantitative information about the physical properties of complex biological molecules without ensemble artifacts obscuring the measurements. We demonstrate computational techniques which aim at more fully utilizing the wealth of information contained in noisy experimental time series. The "noise" comes from multiple sources, e.g. inherent thermal motion, instrument measurement error, etc. The primary focus of this article is a methodology for using time domain based methods for extracting the effective molecular friction from single-molecule pulling data. We studied molecules composed of 8 tandem repeat titin I27 domains, but the modeling approaches have applicability to other single-molecule mechanical studies. The merits and challenges associated with applying such a computational approach to existing single-molecule manipulation data are also discussed.Item Detecting the Biopolymer Behavior of Graphene Nanoribbons in Aqueous Solution(Springer Nature, 2016) Wijeratne, Sithara S.; Penev, Evgeni S.; Lu, Wei; Li, Jingqiang; Duque, Amanda L.; Yakobson, Boris I.; Tour, James M.; Kiang, Ching-Hwa; Richard E. Smalley Institute for Nanoscale Science & TechnologyGraphene nanoribbons (GNR), can be prepared in bulk quantities for large-area applications by reducing the product from the lengthwise oxidative unzipping of multiwalled carbon nanotubes (MWNT). Recently, the biomaterials application of GNR has been explored, for example, in the pore to be used for DNA sequencing. Therefore, understanding the polymer behavior of GNR in solution is essential in predicting GNR interaction with biomaterials. Here, we report experimental studies of the solution-based mechanical properties of GNR and their parent products, graphene oxide nanoribbons (GONR). We used atomic force microscopy (AFM) to study their mechanical properties in solution and showed that GNR and GONR have similar force-extension behavior as in biopolymers such as proteins and DNA. The rigidity increases with reducing chemical functionalities. The similarities in rigidity and tunability between nanoribbons and biomolecules might enable the design and fabrication of GNR-biomimetic interfaces.Item DNA Free Energy Landscapes and RNA Nano-Self-Assembly Using Atomic Force Microscopy(2014-03-26) Frey, Eric William; Kiang, Ching-Hwa; Deem, Michael W.; Ajayan, Pulickel M.There is an important conceptual lesson which has long been appreciated by those who work in biophysics and related interdisciplinary fields. While the extraordinary behavior of biological matter is governed by its detailed atomic structure and random fluctuations, and is therefore difficult to predict, it can nevertheless be understood within simplified frameworks. Such frameworks model the system as consisting of only one or a few components, and model the behavior of the system as the occupation of a single state out of a small number of states available. The emerging widespread application of nanotechnology, such as atomic force microscopy (AFM), has expanded this understanding in eye-opening new levels of detail by enabling nano-scale control, measurement, and visualization of biological molecules. This thesis describes two independent projects, both of which illuminate this understanding using AFM, but which do so from very different perspectives. The organization of this thesis is as follows. Chapter 1 begins with an experimental background and introduction to AFM, and then describes our setup in both single-molecule manipulation and imaging modes. In Chapter 2, we describe the first project, the motivation for which is to extend methods for the experimental determination of the free energy landscape of a molecule. This chapter relies on the analysis of single-molecule manipulation data. Chapter 3 describes the second project, the motivation for which is to create RNA-based nano-structures suitable for future applications in living mammalian cells. This chapter relies mainly on imaging. Chapters 2 and 3 can thus be read and understood separately.Item DNA under Force: Mechanics, Electrostatics, and Hydration(MDPI AG, 2015) Li, Jingqiang; Wijeratne, Sithara S.; Qiu, Xiangyun; Kiang, Ching-HwaQuantifying the basic intra- and inter-molecular forces of DNA has helped us to better understand and further predict the behavior of DNA. Single molecule technique elucidates the mechanics of DNA under applied external forces, sometimes under extreme forces. On the other hand, ensemble studies of DNA molecular force allow us to extend our understanding of DNA molecules under other forces such as electrostatic and hydration forces. Using a variety of techniques, we can have a comprehensive understanding of DNA molecular forces, which is crucial in unraveling the complex DNA functions in living cells as well as in designing a system that utilizes the unique properties of DNA in nanotechnology.Item Enhanced Sampling Method in Statistical Physics and Large-Scale Molecular Simulation of Complex Systems(2014-04-25) Zang, Tianwu; Ma, Jianpeng; Kiang, Ching-Hwa; Raphael, Robert M.In large-scale complex systems, traditional computational methods in equilibrium statistical mechanics such as Monte Carlo simulation and molecular dynamics in canonical ensemble often face the broken ergodicity issue, which highly reduces the performance and accuracy of simulation. The past decades have witnessed the development of generalized ensemble, which has significantly enhanced the efficiency of molecular simulation. In this thesis, we get a review of typical generalized ensembles, such as multi-canonical ensemble, parallel tempering, simulating tempering and continuous simulated tempering (CST). We also present a method called parallel continuous simulated tempering(PCST) for enhanced sampling in studying large complex. It mainly inherits and CST method in previous work, while adopts the spirit of parallel tempering, by employing multiple copies with different temperature distributions. The sampling efficiency of PCST was tested in two-dimensional Ising model, Lennard-Jones liquid and all-atom folding simulation of a small globular protein trp-cage in explicit solvent. The results demonstrate that the PCST method has significantly improved sampling efficiency compared with other methods and it is particularly effective in simulating systems with long relaxation time or correlation time.Item Evaluation of Valvular Endothelial Cell Hemostatic Behavior in Native Valves and Novel Co-culture Models(2014-12-03) Balaoing, Liezl Rae; Grande-Allen, Kathryn J; Moake, Joel L; Kiang, Ching-Hwa; Qutub, Amina; Harrington, DanielThe endothelial cell-mediated process of hemostasis is critical in all living heart valve tissues. As these tissues undergo changes with age and disease, the ability for valvular endothelial cells (VECs) to manage anti- and pro-thrombotic mechanisms may also change. Furthermore, degeneration- and thrombosis-related failures in artificial valves emphasizes the need to understand the anti-thrombotic mechanisms of VECs in order to develop effective strategies to endothelialize implants and tissue-engineered heart valves. Therefore, a study was performed to evaluate the regulation and function of von Willebrand Factor (VWF), ADAMTS-13 (VWF cleaving enzyme), and other thrombotic and anti-thrombotic mediators secreted from VECs from different aged valves. This work identified age-related differences in VEC hemostatic protein regulation, and an increased capacity of specific proteins to aggregate within regions of elderly valves, which are known to have age-associated loss of extracellular matrix (ECM) organization that are linked to calcific aortic valve disease. With the knowledge that ECM can influence hemostasis, we then studied changes in VEC hemostatic regulation using synthetic culture conditions that modulated substrate stiffness and adhesive ligands. RKRLQVQLSIRT (RKR), a syndecan binding cell adhesive peptide derived from laminin-α1 G-domain, was optimal for promoting strong VEC adhesion and balanced hemostatic function on hydrogel constructs of various stiffness in comparison to the commonly used integrin binding peptide RGDS. Next, to evaluate interactions between valve cells, magnetic levitation technology was used to co-culture VECs with valvular interstitial cells (VICs) in a 3D scaffoldless aortic valve co-culture (AVCC). The cell-based AVCC design allowed for synthesis of multiple constructs within a few hours. AVCCs had regional localization of CD31 positive VECs at construct surface. Cells in the AVCC interior (including VECs) expressed low levels of α-smooth muscle actin (αSMA), suggesting maintenance of quiescent VIC phenotype, but potential endothelial to mesenchymal differentiation in interior-localized VECs. In addition, AVCCs produced ECM and expressed hemostatic proteins such as endothelial nitric oxide synthase (eNOS) and VWF. In light of the VEC localization within the AVCC potentially affecting healthy phenotype, a more physiologically organized and customizable scaffold model was needed for further evaluation of direct interactions between VECs and VICs. Therefore, previous RKR-functionalization work was combined with strategies for VIC encapsulation in biofunctionalized-MMP degradable hydrogels to develop a 3D adhesive ligand localized hydrogel scaffold for an endothelialized aortic valve co-culture model. The resulting hydrogel-based endothelialized aortic valve model (HEAVM) promoted the formation of a stable VEC monolayer at the scaffold surface, and supported the maintenance of VIC quiescent phenotypes within the scaffold, thereby mimicking physiological valve cell organization in aortic valves. Platelet adhesion and nitric oxide functional assays confirmed healthy VEC cell behavior, while immunohistochemistry and qRT-PCR were used to asses VIC and VEC phenotype and extracellular matrix (ECM) production. Overall, by utilizing principles from cell and extracellular matrix biology, biomechanics, and biomaterials, this work was able to improve the understanding of the VEC roles in valve homeostasis and the pathogenesis of valvular disease. Furthermore, new biomaterial-based models were designed to enhance the field’s understanding of VEC functions and communication with VICs. The knowledge learned from these models may be applied to future evaluation of various valve diseases, as well as endothelialization strategies for valve implants.Item Experimental Free Energy Landscape Reconstruction of DNA Unstacking Using Crooks Fluctuation Theorem(2013-06-05) Frey, Eric; Kiang, Ching-Hwa; Deem, Michael W.; Nordlander, Peter J.Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.Item Force Activation of a Multimeric Adhesive Protein through Domain Conformational Change(2013-07-24) Wijeratne, Sitara; Kiang, Ching-Hwa; Tao, Yizhi Jane; Kono, JunichiroThe force-induced activation of adhesive proteins such as von Willebrand Factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.Item Forces unveil physics in biological systems via atomic force microscopy: from single molecules to single cells(2018-06-08) Li, Jingqiang; Kiang, Ching-HwaForce plays an essential role in many biological systems at different length. Physical forces, together with chemical signals contribute to the proper functioning of various biological processes. For example, cells sense and transduce environmental physical cues into biochemical signals so as to realize different cellular processes, such as proliferation, migration. and apoptosis. Thus elucidating the details of force involved in various biological systems is thus crucial for a complete understanding of their biological mechanisms such as biomolecule's mechanical properties, dynamic conformations, native structure, and cell's physical properties. The ability of probing and studying the forces in biology has been revolutionized over the past two decades. Atomic force microscopy (AFM), for example, has been proven a powerful technique in measuring forces in piconewton range, relevant in biological scales. In this thesis, I explored the AFM application on different biological systems ranging from single molecule level to single cell level. In the first part, I will show that single molecule manipulation by AFM can reveal the mechanical properties, equilibrium states, and dynamic conformations of proteins and nucleic acids. In the second part, I will extend the application of AFM on single cells and show that the cancer cells adaption to microenvironment can be revealed by force signatures. In addition, I will also demonstrate the investigation of invasiveness of cancer cells via a specific cell line using AFM force studies. Finally, I will discuss future outlooks of AFM cell studies.Item Low Resolution ab initio Phasing Method by Modification of Density and Phase in Real and Reciprocal Space(2013-09-30) Liao, Yunxiang; Ma, Jianpeng; Raphael, Robert M.; Kiang, Ching-HwaPhasing problem in X-ray structure determination can be challenging. Several methods, such as Isomorphous Replacement and Molecule Replacement, are frequently used. But their success relies on the availability of either an isomorphous heavy-atom derivative or a high identity homologous model. In this thesis, a low resolution ab initio phasing method is proposed. A large number of trial phases value and their corresponding high density masks are generated and modified alternately in reciprocal and real space. Then, the output phase sets are averaged to give the estimated phases and figure of merit which are capable of capturing key feature of the molecules’ low resolution envelope by Fourier synthesis. Smoothed particle hydrodynamics is employed to animate the high density masks’ modification process. The method is tested and compared with Lunin’s connectivity-based phasing method which also takes advantage of geometric properties of high density masks.Item Measurement of Magnetoplasmon Resonance in a High-Mobility 2-Dimensional Dot Array using a Micro-calorimeter(2013-07-16) Ball, Jason; Du, Rui-Rui; Kiang, Ching-Hwa; Foster, MatthewWe used a calorimetric method to measure the microwave absorption of a high-mobility 2-dimensional electron gas in small magnetic fields. The calorimeter responded well to the signal, even when reduced to a single dot. Curiously, in the smaller dot lattices we found the zero-field plasmon resonances to be much less the than expected values; however a second, larger sample yielded better values. As a result of the high mobility of the sample we also saw evidence of the second harmonic of the plasmon frequency coupling with the cyclotron resonance, although a corresponding edge mode was not found. The calorimetric method appears to be a valid means of measuring signal absorption in a 2DEG and has potential for probing other types of systems.Item Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change(American Physical Society, 2013) Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-HwaThe mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.Item Nanoscale manipulation and studies of individual biomolecules and DNA-based nanostructures(2009) Harris, Nolan C.; Kiang, Ching-HwaNanoscale manipulation of individual biomolecules, using such techniques as the atomic force microscope (AFM) and laser optical tweezers (LOT), has increased the scope and detail with which important biological interactions, such as protein folding, receptor-ligand binding, and double-stranded DNA melting, can be studied. In recent years, single molecule manipulation via AFM has been used to characterize the mechanical properties of various proteins. However, since single molecule manipulation experiments are typically performed under nonequilibrium conditions, extracting thermodynamic properties from these measurements has proven difficult. The derivation of Jarzynski's equality, which relates nonequilibrium work fluctuations to equilibrium free energy differences, provides the possibility for extracting equilibrium information from single molecule manipulation data. Here, single molecule force measurements of the stretching and unfolding of the titin I27 protein are analyzed using Jarzynski's equality to reconstruct the underlying free energy landscape associated with this process. We describe the procedures for the automated selection, alignment, and Jarzynski analysis of single molecule data. We use the recovered equilibrium free energy landscape to estimate thermodynamic properties such as the unfolding free energy barrier, which is in good agreement with estimates from bulk kinetics studies and various simulations. Also, the convergence behavior of Jarzynski's equality with respect to pulling velocity is studied experimentally. We demonstrate that with enough pulling trajectories, Jarzynski's equality will indeed recover the equilibrium free energy landscape for a given process. The number of trajectories required to recover the equilibrium free energy for a given pulling velocity is used to quantify this convergence behavior and identify a range of pulling velocities that is most efficient for thermodynamic analysis of single molecule manipulation data. Single molecule manipulation is also used to characterize DNA melting transitions by repeatedly stretching and relaxing an individual λ-DNA molecule. Here, a force induced transition between B form DNA ( B -DNA) and S form DNA ( S -DNA), prior to dsDNA melting, is observed. The mechanical properties of the various conformations, B -DNA, S -DNA, and single-stranded DNA, are quantified using polymer elasticity models, and are shown to agree well with expectations from previous experiments and theory. Fabrication of DNA-based nanostructures, particularly DNA-gold nanoparticle assemblies, has generated significant interest due to their interesting optical and phase transition properties. Here, the effects of various types of disorder within the DNA-gold nanoparticle system are studied and quantified. We show that the stability of these complex fluids can not be quantified using expectations from free DNA hybridization. For example, when linking pairs of gold nanoparticle probes using a DNA linker sequence, a lack of base-pairing symmetry between the probes creates a disorder that decreases the overall stability of the system. This occurs despite the energy contribution that is gained by adding a single base pair to one probe, creating the lack of symmetry. The assumption that base-pairing defects will lower the stability of the nanoparticle aggregates due a loss of hybridization energy is also found to be violated with surprising frequency. In some cases, nonspecific binding between the gold particle surface and a free DNA base can result in a system with increased stability, despite the loss in energy resulting from a mismatched or deleted base. These observations demonstrate that the DNA interactions within these nanostructures are highly complex and that the system stability is not always governed by free DNA hybridization.Item Quantifying DNA Melting Transitions Using Single-Molecule Force Spectroscopy(2008-09) Calderon, Christopher P.; Chen, Wei-Hung; Lin, Kuan-Jiuh; Harris, Nolan C.; Kiang, Ching-HwaWe stretched a DNA molecule using atomic force microscope and quantified the mechanical properties associated withﾠBandﾠSﾠforms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA (ssDNA). We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.Item Quantifying Multiscale Noise Sources in Single-Molecule Time Series(2008-09) Calderon, Christopher P.; Harris, Nolan C.; Kiang, Ching-Hwa; Cox, Dennis D.When analyzing single-molecule data, a low-dimensional set of system observables typically serve as the observational data. We calibrate stochastic dynamical models from time series that record such observables. Numerical techniques for quantifying noise from multiple time-scales in a single trajectory, including experimental instrument and inherent thermal noise, are demonstrated. The techniques are applied to study time series coming from both simulations and experiments associated with the nonequilibrium mechanical unfolding of titin's I27 domain. The estimated models can be used for several purposes: (1) detect dynamical signatures of "rare events" by analyzing the effective diffusion and force as a function of the monitored observable, (2) quantify the influence that conformational degrees of freedom, which are typically difficult to directly monitor experimentally, have on the dynamics of the monitored observable, (3) quantitatively compare the inherent thermal noise to other noise sources, e.g. instrument noise, variation induced by conformational heterogeneity, etc., (4) simulate random quantities associated with repeated experiments, (5) apply pathwise, i.e. trajectory-wise, hypothesis tests to assess the goodness-of-fit of the models and even detect conformational transitions in noisy signals. These items are all illustrated with several examples.Item Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix(Elsevier, 2016) Wijeratne, Sithara S.; Martinez, Jerahme R.; Grindel, Brian J.; Frey, Eric W.; Li, Jingqiang; Wang, Liyun; Farach-Carson, Mary C.; Kiang, Ching-HwaPerlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170 ± 20 nm and a diameter of 2–4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young's modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.Item Single Molecule Force Signatures in Biological Physics(2015-11-11) Wijeratne, Sithara Suransi; Kiang, Ching-HwaSingle molecule manipulation has opened up new research frontiers in understanding how biology and medicine function at the microscopic level. Quantitative information on the structure, conformation and dynamics of biological molecules can be revealed by the single molecule force measurements. Recently single molecule manipulation via the atomic force microscope (AFM) has been used to characterize the mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which is essential in initiating platelet adhesion. The mechanical force-induced functional change of VWF plays a crucial role in hemostasis, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We also investigated the effect of high fluid shear stress on soluble dimeric VWF (DVWF). DVWF is the smallest unit that polymerizes to construct large VWF multimers. Our data indicate that, unlike PVWF multimers, DVWF is not altered by high shear stress. We conclude that DVWF is not capable of self-association under shear into a conformation analogous to that attained by sheared large VWF multimers. Single molecule force signatures were also used to characterize the mechanical properties of proteins related to the complement system and the extracellular matrix. Beyond the investigation of proteins, we applied this technique to understand the mechanical behavior of graphene nanoribbons, a potential biomaterial, which revealed a biopolymer behavior. Finally, the AFM technique can be extended from probing single molecules to capturing characteristics of the whole cell. These single cell experiments revealed the forces related to pulling tethers from the cell membrane.Item Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor(American Physical Society, 2016) Wijeratne, Sithara S.; Li, Jingqiang; Yeh, Hui-Chun; Nolasco, Leticia; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-HwaVon Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100dynes/cm2 for 3 min at 37∘C). We conclude that under the shear conditions used (100dynes/cm2 for 3 min at 37∘C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.Item Understanding the physics of DNA using nanoscale single-molecule manipulation(Springer, 2012) Frey, Eric W.; Gooding, Ashton A.; Wijeratne, Sitara; Kiang, Ching-HwaProcesses for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code.