Bulletin of the American Physical Society
APS March Meeting 2016
Volume 61, Number 2
Monday–Friday, March 14–18, 2016; Baltimore, Maryland
Session V41: Physics of Proteins: Mechanics and ForcesFocus
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Sponsoring Units: DBIO DPOLY DCOMP Chair: Tom Chou, University of California, Los Angeles Room: 344 |
Thursday, March 17, 2016 2:30PM - 3:06PM |
V41.00001: Rationally designing the mechanical properties of protein hydrogels Invited Speaker: Yi Cao Naturally occurring biomaterials possess diverse mechanical properties, which are critical to their unique biological functions. However, it remains challenging to rationally control the mechanical properties of synthetic biomaterials. Here we provide a bottom-up approach to rationally design the mechanical properties of protein-based hydrogels. We first use atomic fore microscope (AFM) based single-molecule force spectroscopy to characterize the mechanical stability of individual protein building blocks. We then rationally design the mechanical properties of hydrogels by selecting different combination of protein building blocks of known mechanical properties. As a proof-of-principle, we demonstrate the engineering of hydrogels of distinct extensibility and toughness. This simple combinatorial approach allows direct translation of the mechanical properties of proteins from the single molecule level to the macroscopic level and represents an important step towards rationally designing the mechanical properties of biomaterials. [Preview Abstract] |
Thursday, March 17, 2016 3:06PM - 3:18PM |
V41.00002: Nonlinear elasticity of disordered fiber networks Jingchen Feng, Herbert Levine, Xiaoming Mao, Leonard M. Sander One of the most striking mechanical properties in disordered biopolymer gels is strong nonlinearities. In the case of athermal gels (such as collagen- I) the nonlinearity has long been associated with a crossover from a bending dominated to a stretching dominated regime of elasticity. The physics of this crossover is related to the existence of a central-force isostatic point and to the small bending modulus for most gels. This crossover induces scaling behavior for the elastic moduli. In particular, for linear elasticity such a scaling law has been demonstrated by Broedersz et al. We generalize the scaling to the nonlinear regime with a two-parameter scaling law involving three critical exponents. We do numerical testing of the scaling law for two disordered lattice models, and find a good scaling collapse for the shear modulus in both the linear and nonlinear regimes. We compute all the critical exponents for the two lattice models and discuss the applicability of our results to real systems. [Preview Abstract] |
Thursday, March 17, 2016 3:18PM - 3:30PM |
V41.00003: Mechanosensing by tethered membrane channels Benedikt Sabass, Howard A. Stone Force-gated membrane channels are a paradigm of biological mechanosensing. These channels are often tethered to cytoskeletal elements, which allows direct transmission of the mechanical signal. How force at tethers leads to channel opening is unknown. Here, we focus on the generic role of membrane-channel interaction for gating. We propose a scaling relation, linking protein deformation under force to membrane energy. A minute conical deformation during gating leads to an elastic energy gain that far exceeds the thermal energy. Force thresholds for gating are in the experimentally inferred range and are robust against changes of membrane tension. We also study a detailed model for membrane-mediated interactions among channels. In general, interactions reduce the force threshold, leading to cooperatively enhanced gating. [Preview Abstract] |
Thursday, March 17, 2016 3:30PM - 3:42PM |
V41.00004: Group transfer theory of single molecule imaging experiments in the F-ATPase biomolecular motor Sandor Volkan-Kacso, Rudolph Marcus I describe a chemo-mechanical theory to treat single molecule imaging and ``stalling'' experiments on the F-ATPase enzyme. This enzyme is an effective stepping biomolecular rotary motor with a rotor shaft and a stator ring. Using group transfer theoretical approach the proposed structure-based theory couples the binding transition of nucleotides in the stator subunits and the physics of torsional elasticity in the rotor. The twisting of the elastic rotor domain acts as a perturbation upon the driving potential, the Gibbs free energy. In the theory, without the use of adjustastable parameters, we predict the rate and equilibrium constant dependence of steps such as ATP binding and phosphate release as a function of manipulated rotor angle. Then we compare these predictions to available data from stalling experiments. Besides treating experiments, the theory can provide guides for atomistic simulations, which could calculate the reorganization parameter and the torsional spring constant. The framework is generic and I discuss its application to other single molecule experiments, such as controlled rotation and other biomolecular motors, including motor-DNA complexes and linear motors.[PNAS, Early Edition, Oct. 19, 2015, doi: 10.1073/pnas.1518489112] [Preview Abstract] |
Thursday, March 17, 2016 3:42PM - 3:54PM |
V41.00005: Fluorescent ATP analog mant-ATP reports dynein activity in the isolated \textit{Chlamydomonas} axoneme Maria Feofilova, Jonathon Howard Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme. [Preview Abstract] |
Thursday, March 17, 2016 3:54PM - 4:06PM |
V41.00006: Limiting Speed of the Bacterial Flagellar Motor Jasmine Nirody, Richard Berry, George Oster The bacterial flagellar motor (BFM) drives swimming in a wide variety of bacterial species, making it crucial for several fundamental biological processes including chemotaxis and community formation. Recent experiments have shown that the structure of this nanomachine is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we put forward a model for the torque generation mechanism of this motor and propose that the maximum speed of the motor increases as additional torque-generators are recruited. This is contrary to the current widely-held belief that there is a universal upper limit to the speed of the BFM. Our result arises from the assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads. [Preview Abstract] |
Thursday, March 17, 2016 4:06PM - 4:18PM |
V41.00007: A mechanochemical model for myosin VI Riina Tehver, Amanda Jack, Ian Lowe Myosin VI is a motor protein that transports cellular cargo along actin filaments. This transport takes place as a result of a coordinated mechano-chemical cycle that is controlled by external variables including imposed force and nucleotide concentrations.~ We present a model that captures the different dynamic pathways that myosin VI can take in response to these variables.~ The results of our model for experimentally observable quantities, such as the motor velocity or run length, agree with available experimental data, and we can also make predictions beyond the tested regimes.~ Using the model, we study how myosin VI reacts to its environment and test its operational efficiency. [Preview Abstract] |
Thursday, March 17, 2016 4:18PM - 4:30PM |
V41.00008: Talin mediated force transmission and mechanosensing Jie Yan, Mingxi Yao, Benjamin Goult, Michael Sheetz Cells adhere to extracellular matrix (ECM) through focal adhesion. Talin is a cytoplasmic adapter protein that links the actin cytoskeleton to focal adhesion, playing a central role in regulation of cell spreading and migration. Talin's functions depend on the binding of talin rod domains to a cytoplasmic protein vinculin in a force dependent manner. By stretching full-length talin rod using magnetic tweezers, we have determined the force-dependent unfolding and refolding rates of subdomains in talin rod. Kinetics simulations based on these rates have revealed that talin rod can serve as a force buffer, capable of maintaining tension in talin in a range of 5-10 pN over a wide range of extension change of talin rod from 50 nm to 400 nm. Further, this level of force is found able to expose the cryptic vinculin-binding sites, promoting subsequent binding of the head domain of vinculin with a nano Molar affinity. Such a force-sensitive interaction between talin rod and vinculin is described by a force-dependent dissociation constant derived based on the mechanical stability of the talin rod domains. Together, these results provide important insights into the mechanosensing at focal adhesion that is crucial for cells to sense and respond to their microenvironments. [Preview Abstract] |
Thursday, March 17, 2016 4:30PM - 4:42PM |
V41.00009: Directly measuring single molecule heterogeneity in proteins and RNA using force spectroscopy Michael Hinczewski, Changbong Hyeon, Devarajan Thirumalai One of the most intriguing results of single molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with stochastic interconversions occurring only at macroscopic timescales, fractions of a second or longer. Though we now have proof of functional heterogeneity in a handful of systems---enzymes, motors, adhesion complexes---identifying and measuring it remains a formidable challenge. We show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single molecule techniques: AFM or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Our work suggests experimental approaches for estimating the timescales of these fluctuations with unprecedented accuracy. [Preview Abstract] |
Thursday, March 17, 2016 4:42PM - 4:54PM |
V41.00010: Bayesian Uncertainty Quantification for Bond Energies and Mobilities Using Path Integral Analysis Pak-Wing Fok, Joshua Chang, Tom Chou Dynamic single-molecule force spectroscopy is often used to distort bonds. The resulting responses, in the form of rupture forces and trajectories of displacements, are used to reconstruct bond potentials. Such approaches often rely on simple parameterizations of one-dimensional bond potentials and/or large amounts of trajectory data. Parametric approaches typically fail at inferring complicated bond potentials with multiple minima, while piecewise estimation may not guarantee smooth results. Existing techniques also do not address spatial variations in the diffusivity that may arise from inhomogeneous coupling to other degrees of freedom in the macromolecule. To address these challenges, we develop an empirical Bayesian approach that incorporates data and regularization terms into a path integral. All experimental and statistical parameters in our method are estimated from the data. Upon testing our method on simulated data, our regularized approach requires less data and allows simultaneous inference of both complex bond potentials and diffusivities. We show that the accuracy of the reconstructed bond potential is sensitive to the spatially varying diffusivity and accurate reconstruction can be expected only when both are simultaneously inferred. [Preview Abstract] |
Thursday, March 17, 2016 4:54PM - 5:06PM |
V41.00011: Base-by-Base Counting of Nucleotide Incorporations by DNA Polymerase Mackenzie W. Turvey, O. Tolga Gul, Kaitlin M. Pugliese, Denys O. Marushchak, Arith J. Rajapakse, Gregory A. Weiss, Phillip G. Collins Previously, the catalytic cycle of DNA polymerase has been recorded by tethering single polymerase molecules to single-walled carbon nanotube field effect transistors (FETs) [1]. As the polymerase incorporates nucleotides into a single-stranded DNA template, it generates electrical signals in the SWCNT-FET. Here, we investigate the accuracy of this electronic method by using low concentrations (\textless 10 nM) of DNA template, such that the signal consists of long, diffusion-limited pauses interrupted by template binding and a burst of nucleotide incorporation events. By counting the events generated by as few as 10 template molecules, template length has been correctly determined with \textless 1 base pair resolution. Furthermore, differing template lengths can be identified and correctly enumerated in solutions containing mixtures of templates. Processivity of the Klenow Fragment of DNA polymerase currently limits read lengths to 50-100 base pairs, but the FET technique should work equally well with longer-processivity polymerases. 1. T.J. Olsen, et. al., ``Electronic Measurements of Single-Molecule Processing by DNA polymerase I (Klenow fragment),'' JACS 135, 7855 (2013). [Preview Abstract] |
Thursday, March 17, 2016 5:06PM - 5:18PM |
V41.00012: Sequence and Structure Dependent DNA-DNA Interactions Benjamin Kopchick, Xiangyun Qiu Molecular forces between dsDNA strands are largely dominated by electrostatics and have been extensively studied. Quantitative knowledge has been accumulated on how DNA-DNA interactions are modulated by varied biological constituents such as ions, cationic ligands, and proteins. Despite its central role in biology, the sequence of DNA has not received substantial attention and ``random'' DNA sequences are typically used in biophysical studies. However, \textasciitilde 50{\%} of human genome is composed of non-random-sequence DNAs, particularly repetitive sequences. Furthermore, covalent modifications of DNA such as methylation play key roles in gene functions. Such DNAs with specific sequences or modifications often take on structures other than the canonical B-form. Here we present series of quantitative measurements of the DNA-DNA forces with the osmotic stress method on different DNA sequences, from short repeats to the most frequent sequences in genome, and to modifications such as bromination and methylation. We observe peculiar behaviors that appear to be strongly correlated with the incurred structural changes. We speculate the causalities in terms of the differences in hydration shell and DNA surface structures. [Preview Abstract] |
Thursday, March 17, 2016 5:18PM - 5:30PM |
V41.00013: \textit{In vivo }Studies of VEGFR2 Interactions in the Presence and Absence of VEGF Christopher King, Dr. Kalina Hristova Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a receptor tyrosine kinase (RTK) that is critical for vasculogenesis and angiogenesis.~ Enhanced VEGFR2 signaling is often correlated with malignancy.~ ~Recently, it was shown that full-length VEGFR2 exists in a monomer-dimer equilibrium in the absence of bound VEGF.~~ Thus, the canonical model of RTK activation does not seem to adequately describe the behavior of VEGFR2 in the cell membrane.~ In order to understand the role that VEGFR2 extracellular domain plays in unliganded dimerization in live cells, we utilize \textbf{F}ully Quantified \textbf{S}pectral \textbf{I}maging (FSI) to probe the interactions of VEGFR2 mutant constructs with rationally truncated EC domains.~~ In addition, we investigate the stoichiometry of ligand binding to VEGFR2 EC domain as a function of~ VEGF concentration and total receptor expression.~ [Preview Abstract] |
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