Bulletin of the American Physical Society
Annual Meeting of the APS Four Corners Section
Volume 62, Number 17
Friday–Saturday, October 20–21, 2017; Fort Collins, CO
Session E6: Biophysics I |
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Chair: Vern Hart, Utah Valley Unversity Room: Lory Student Center 308 |
Friday, October 20, 2017 1:20PM - 1:44PM |
E6.00001: Structure and function of electrogenic sodium/proton antiporter membrane proteins Invited Speaker: Oliver Beckstein Sodium/proton antiporters are integral membrane proteins that are vital for cell homeostasis. In bacteria, they pump sodium ions out of the cell and enable survival in high-salt environments. In humans, they maintain cellular pH and their dysfunction is linked to a variety of complex diseases, including cancer, cardiovascular pathophysiology, and autism. Na$^+$/H$^+$ antiporters are secondary active transporters that utilize the electrochemical gradient of one ionic species to drive the energetically uphill transmembrane transport of the other species. They operate by the alternating access mechanism whereby the protein cycles between an outward facing and inward facing conformation to switch the exposure of substrate binding sites between the extracellular and the intracellular environment. We used a combination of molecular dynamics simulations with X-ray crystallography and functional measurements to address key questions about the transport mechanism in the two electrogenic bacterial antiporters NhaA and NapA, which both exchange 2 H$^+$ for 1 Na$^+$. In particular, the likely sodium and proton binding sites overlap, as predicted by a previously proposed competitive binding mechanism. A large ``elevator-like'' conformational transition moves the Na$^+$ binding site across the membrane, consistent with the alternating access mechanism. Based on simulations with varying protonation states of conserved ionizable residues and explicit constant pH MD simulations, we put forward a detailed hypothesis for the transport mechanism. Two protons are carried by a conserved aspartate and a conserved lysine residue. The Na+ binding site is formed by two conserved aspartate residues. The protonated lysine forms a salt bridge with the aspartate that does not carry a proton. Binding of Na$^+$ disrupts the salt bridge and facilitates the release of the proton from the lysine, thus maintaining the experimentally observed competitive binding mechanism. The binding site itself is translocated in a rigid fashion across the membrane, consistent with the alternating access mechanism. Functional transport measurements of mutants support the hypothesis that the Asp-Lys salt bridge is required for electrogenic transport. [Preview Abstract] |
Friday, October 20, 2017 1:44PM - 1:56PM |
E6.00002: Mechanisms of selective transport through nuclear pore complex mimics Laura Maguire, Michael Stefferson, Katherine Rainey, Nathan Crossette, Eric Verbeke, Meredith Betterton, Loren Hough Few cellular processes require such intricate active control as transport through the nuclear envelope. The nuclear pore complex (NPC) facilitates all transport, preventing most macromolecules from crossing the envelope while allowing the passage of transport factors (TFs) and their cargo. While the basic biochemical interactions of transport are well-understood, the detailed mechanism remains a topic of significant debate. We create tunable mimics of the NPC using PEG hydrogels filled with FG nucleoporins (FG nups), the intrinsically disordered proteins that line the NPC channel in vivo. We also model transport using reaction-diffusion equations. The results suggest that (1) the flexible nature of the disordered FG nups and (2) the transient, multivalent nature of FG nup -- TF interactions are together sufficient for selectivity. Our model makes selectivity predictions that will be directly testable in our experimental setup. We aim to use the model to tune the mimic's parameters to maximize selectivity. [Preview Abstract] |
Friday, October 20, 2017 1:56PM - 2:08PM |
E6.00003: Application of Maximum Caliber to Infer Rates in Genetic Circuits Taylor Firman, Kingshuk Ghosh Learning the underlying details of a gene network is a major challenge in cellular and synthetic biology. We address this challenge by building a chemical kinetic model that utilizes information encoded in the stochastic protein expression trajectories typically measured in experiments. The applicability of the proposed method is demonstrated in an auto-activating genetic circuit, a common motif in natural and synthetic gene networks. Our approach is based on the principle of Maximum Caliber (MaxCal) -- a dynamical analogue of the principle of maximum entropy -- and builds a minimal model using only three constraints: i) protein synthesis, ii) protein degradation, and iii) positive feedback. The MaxCal model was benchmarked against synthetic data generated using a Gillespie algorithm on a known reaction network. MaxCal accurately predicts underlying rate parameters of protein synthesis and degradation as well as experimental observables such as protein number and dwell time distributions. This `top-down' methodology based on minimal information -- in contrast to traditional `bottom-up' approaches that require ad-hoc knowledge of circuit details -- provides a powerful tool to accurately infer underlying details of feedback circuits that are not otherwise visible in experiments. [Preview Abstract] |
Friday, October 20, 2017 2:08PM - 2:20PM |
E6.00004: Mitochondrial fluctuations as a measure of active biomechanical properties of mammalian cells Wenlong Xu, Elaheh Alizadeh, Jordan Castle, Ashok Prasad A single-cell assay of mechanical properties would give significant insights into cellular processes. Force spectrum microscopy is one such technique, which involves both active and passive particle tracking microrheology on the same cells. Since active microrheology requires expensive instruments, it is of great interest to develop simpler alternatives. Here we study an alternative using endogenous mitochondrial fluctuations, rather than fluorescent beads, in particle tracking microrheology. Mitochondria of the C3H-10T1/2 cell line are labeled and tracked using confocal microscopy, their mean square displacement (MSD) measured, and mechanical parameters calculated. We found that the MSD of mitochondria resembles that of particles in viscoelastic media. However, comparisons of MSD between controls and cells disrupted in the actin or microtubule network showed surprisingly small effects, while ATP-depleted cells showed significantly decreased MSD, and characteristics of thermally driven fluctuations. Active fluctuations are distinguished from passive fluctuations by treatment with ATP synthesis inhibitors, both of which were fed into the determination of force spectrum. With similar results to published studies, this method is potentially very useful due to its simplicity. [Preview Abstract] |
Friday, October 20, 2017 2:20PM - 2:32PM |
E6.00005: BioPhysical Applications of Non-Equilibrium Dynamics of Flexible Self Propelled Rods Tyler Thompson, Jeff Moore, Meredith Betterton, Matt Glaser Interactions between the microtubule cytoskeleton and its associated proteins give rise to complex behavior that is essential to many cellular processes, including the self-assembly and organization of the cytoskeleton during mitosis. Experiments to observe microtubule organization have used the directed motor protein kinesin-1 to generate polar forces along microtubules to produce filament-gliding motility assays. At different microtubule densities and ATP concentrations, these active systems exhibit different phenomenological behaviors, such as flocking microtubule bundles and nematic liquid crystal phases. Additionally, steady-state structures that resemble ring-shaped spools of single or bundled filaments appear, which arise due to the finite persistence length of the filaments. These spools have also been observed in previous studies of motility assays with actin and myosin, but their genesis and stability are nevertheless poorly understood. In order to better understand the behavior of these systems quantitatively, we have developed simulations of gliding filaments on motility assays by modeling the filaments as wormlike chains that undergo steric interactions and experience an orientation-dependent force per unit length. In particular, we characterize the phase transition behavior between the isotropic flocking, nematic laning, and spooling phases as a function of filament density, persistence length, and driving. These simulations make predictions for the behavior of future motility assay experiments and can provide additional insights into cytoskeletal dynamics. [Preview Abstract] |
Friday, October 20, 2017 2:32PM - 2:44PM |
E6.00006: Development of a radioactive holmium-166 skin patch to treat skin diseases Martin Ritter, Frederic Sarazin, Jeramy Zimmerman Skin cancer is the most common form of cancer affecting over 3 million people per year in the US alone. The two most common forms of cancer, basal and squamous cell carcinoma, are easily removable if detected early and properly treated. These cancers can become deadly if allowed to spread. There currently exist multiple treatment options with varying success rates such as surgical excision and radiotherapy. However, these methods are not suited to treat sensitive areas such as the eyelids, nose, and lips. We propose a holmium based radioactive skin patch to achieve locally therapeutic radiation doses with no damage to underlying tissues. Holmium-166 is a beta emitter produced by a neutron capture by holmium-165 with a maximum energy of 1.8 MeV and a half-life of 26.84 hours. The half-life of the holmium allows production the patches off-site and the radiation type deposits over 90\% of the dose within 2 mm of skin. We used DC sputtering to deposit a thin film of holmium on a Kapton substrate and characterized the thickness and uniformity of the film with a scanning electron microscope. We activated the holmium at the 1 MW TRIGA reactor located at the Federal Center in Lakewood, CO and achieved activities of up to 1 mCi, an activity within the range for therapeutic treatment. [Preview Abstract] |
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