Bulletin of the American Physical Society
APS March Meeting 2013
Volume 58, Number 1
Monday–Friday, March 18–22, 2013; Baltimore, Maryland
Session Z42: Focus Session: Single Molecule Studies of Protein Nanomachines |
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Sponsoring Units: DBIO Chair: Jing Xu, University of California, Merced Room: Hilton Baltimore Holiday Ballroom 3 |
Friday, March 22, 2013 11:15AM - 11:51AM |
Z42.00001: Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1 Invited Speaker: Jing Xu Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head--tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results (Nat. Commun., 3:754, 2012) provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. [Preview Abstract] |
Friday, March 22, 2013 11:51AM - 12:03PM |
Z42.00002: Investigation of the kinesin stepping mechanism via simulated annealing B.D. Jacobson, S.J. Koch, S.R. Atlas As kinesin processes along the microtubule, the cycle of different chemical states and physical conformations that the protein assumes can be represented by a kinetic model. Such models are preferred for numerical calculations since information about the kinesin stepping mechanism at all levels, from the atomic to the microscopic scale, is fully contained in the particular states of the cycle, in how states transition, and in the rate constants associated to each transition. This greatly simplifies the model of the mechanism while providing a reliable physical picture. We have developed a methodology that optimizes a kinetic model for kinesin built with a minimum of a priori assumptions about the mechanism. We combine Markov chain calculations and simulated annealing optimization to find the rate constants that effectively fit experimental data on kinesin speed and processivity. This optimization scheme leads us to choose the cycle that is most likely to realize the kinesin step. We report details of our kinetic model simulations which best fit experimental data for both single-molecule and gliding motility assays at varying ATP concentrations. [Preview Abstract] |
Friday, March 22, 2013 12:03PM - 12:15PM |
Z42.00003: The Role of Secondary Structure on the Mechanical Properties of Titin Ravi Kappiyoor, Daniel Dudek, Ishwar Puri Elastomeric proteins are characterized by high resilience and low stiffness. Recent work suggests that charge interactions between the proteins and water have a large role in these mechanical properties. However, some elastomeric proteins are nonpolar, and, as such, do not have high charge interactions with the surrounding water. This indicates that there are also other factors at work. We consider the role of secondary structure (i.e. alpha helices and beta sheets) on the mechanical properties of one such elastomeric protein, titin. Molecular dynamics simulations are performed on four different configurations: (i) the PEVK domain of titin (little secondary structure in its natural state), (ii) an immunoglobulin-like domain of titin (high secondary structure in its natural state), (iii) the same immunoglobulin-like domain with all of its secondary structure artificially removed, and finally (iv) the PEVK domain linked to the immunoglobulin-like domain in its natural state. These simulations will provide key insight on the role of secondary structure on mechanical properties, which can be used to more efficiently design smart materials. [Preview Abstract] |
Friday, March 22, 2013 12:15PM - 12:27PM |
Z42.00004: Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles Boqian Yang, Tao He, C\'edric Grauffel, Nathalie Reuter, Mary Roberts, Anne Gershenson Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that \textit{Bacillus thuringiensis} PI-PLC specifically binds to phosphatidylcholine (PC)--rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. [Preview Abstract] |
Friday, March 22, 2013 12:27PM - 1:03PM |
Z42.00005: Single-Molecule Discrimination within Dendritic Spines of Discrete Perisynaptic Sites of Actin Filament Assembly Driving Postsynaptic Reorganization Invited Speaker: Thomas A. Blanpied In the brain, the strength of synaptic transmission between neurons is principally set by the organization of proteins within the receptive, postsynaptic cell. Synaptic strength at an individual site of contact can remain remarkably stable for months or years. However, it also can undergo diverse forms of plasticity which change the strength at that contact independent of changes to neighboring synapses. Such activity-triggered neural plasticity underlies memory storage and cognitive development, and is disrupted in pathological physiology such as addiction and schizophrenia. Much of the short-term regulation of synaptic plasticity occurs within the postsynaptic cell, in small subcompartments surrounding the synaptic contact. Biochemical subcompartmentalization necessary for synapse-specific plasticity is achieved in part by segregation of synapses to micron-sized protrusions from the cell called dendritic spines. Dendritic spines are heavily enriched in the actin cytoskeleton, and regulation of actin polymerization within dendritic spines controls both basal synaptic strength and many forms of synaptic plasticity. However, understanding the mechanism of this control has been difficult because the submicron dimensions of spines limit examination of actin dynamics in the spine interior by conventional confocal microscopy. To overcome this, we developed single-molecule tracking photoactivated localization microscopy (smtPALM) to measure the movement of individual actin molecules within living spines. This revealed inward actin flow from broad areas of the spine plasma membrane, as well as a dense central core of heterogeneous filament orientation. The velocity of single actin molecules along filaments was elevated in discrete regions within the spine, notably near the postsynaptic density but surprisingly not at the endocytic zone which is involved in some forms of plasticity. We conclude that actin polymerization is initiated at many well-separated foci within spines, an organization that may be necessary for the finely tuned adjustment of synaptic molecular content that underlies functional plasticity. Indeed, further single-molecule mapping studies confirm that actin polymerization drives reorganization of molecular organization at the synapse itself. [Preview Abstract] |
Friday, March 22, 2013 1:03PM - 1:15PM |
Z42.00006: Solvated dissipative electro-elastic network model of hydrated proteins Daniel Martin Elastic network models coarse grain proteins into a network of residue beads connected by springs. We add dissipative dynamics to this mechanical system by applying overdamped Langevin equations of motion to normal-mode vibrations of the network. In addition, the network is made heterogeneous and softened at the protein surface by accounting for hydration of the ionized residues. Solvation changes the network Hessian in two ways. Diagonal solvation terms soften the spring constants and off-diagonal dipole-dipole terms correlate displacements of the ionized residues. The model is used to formulate the response functions of the electrostatic potential and electric field appearing in theories of redox reactions and spectroscopy. We also formulate the dielectric response of the protein and find that solvation of the surface ionized residues leads to a slow relaxation peak in the dielectric loss spectrum, about two orders of magnitude slower than the main peak of protein relaxation. Finally, the solvated network is used to formulate the allosteric response of the protein to ion binding. The global thermodynamics of ion binding is not strongly affected by the network solvation, but it dramatically enhances conformational changes in response to placing a charge at the a the active site. [Preview Abstract] |
Friday, March 22, 2013 1:15PM - 1:27PM |
Z42.00007: Single Molecule Electron Paramagnetic Resonance Richelle M. Teeling-Smith, Ezekiel Johnston-Halperin, Michael G. Poirier, P. Chris Hammel Electron paramagnetic resonance (EPR) is a powerful spectroscopic tool for studying the dynamics of biomolecular systems. EPR measurements on bulk samples using a commercial X-band spectrometer provide insight into atomic-scale structure and dynamics of ensembles of biomolecules. Separately, single molecule measurements of biomolecular systems allow researchers to capture heterogeneous behaviors that have revealed the molecular mechanisms behind many biological processes. We are merging these two powerful techniques to perform single molecule EPR$.$ In this experiment, we selectively label double-stranded DNA molecules with nitrogen-vacancy (NV) center nanodiamonds and optically detect the magnetic resonance of the NV probe. Shifts and broadening of our EPR peaks indicate the changing position of the attached DNA relative to the applied magnetic field. Using this new technique, we have successfully measured the first EPR spectrum of a single biomolecule. By controlling the geometry of the diamond and the applied magnetic field, we will quantitatively determine the rotational and translational dynamics of single biomolecules. This research provides the foundation for an advanced single molecule magnetic resonance approach to studies of complex biomolecular systems. [Preview Abstract] |
Friday, March 22, 2013 1:27PM - 1:39PM |
Z42.00008: Turning a Single Molecule into an Electric Motor Charles Sykes Significant progress has been made in the construction of molecular motors powered by light and by chemical reactions, but electrically-driven motors have only just been demonstrated [1,2] after many theoretical proposals. Studying the rotation of molecules bound to surfaces offers the advantage that a single layer can be assembled, monitored and manipulated using the tools of surface science. Thioether molecules constitute a simple, robust system with which to study molecular rotation as a function of temperature, electron energy, applied fields, and proximity of neighboring molecules. A butyl methyl sulphide (BuSMe) molecule adsorbed on a copper surface can be operated as a single-molecule electric motor. Electrons from a scanning tunneling microscope are used to drive directional motion of the BuSMe molecule in a two terminal setup. Moreover, the temperature and electron flux can be adjusted to allow each rotational event to be monitored at the molecular-scale in real time. The direction and rate of the rotation are related to the chiralities of the molecule and the tip of the microscope (which serves as the electrode), which illustrates the importance of the symmetry of the metal contacts in atomic-scale electrical devices. [1] Experimental Demonstration of a Single-Molecule Electric Motor H. L. Tierney, C. J. Murphy, A. D. Jewell, A. E. Baber, E. V. Iski, H. Y. Khodaverdian, A. F. McGuire, Nikolai Klebanov and E. C. H. Sykes - Nature Nanotechnology 2011, 6, 625-629 [2] Electrically driven directional motion of a four-wheeled molecule on a metal surface Kudernac, T., Ruangsupapichat, N., Parschau, M., Macia, B., Katsonis, N., Harutyunyan, S. R., Ernst, K.-H., Feringa, B. L. - Nature 2011, 479, 208--211 [Preview Abstract] |
Friday, March 22, 2013 1:39PM - 1:51PM |
Z42.00009: Characterization of cellular traction forces at the single-molecule level Alexander Dunn The ability of cells to generate and respond to mechanical cues is an essential aspect of stem cell differentiation, embryonic development, and our senses of touch and hearing. However, our understanding of the roles of mechanical force in cell biology remains in its infancy, due largely to a lack of tools that measure the forces generated by living cells at the molecular scale. Here we describe a new technique termed Molecular Force Microscopy (MFM) that visualizes the forces exerted by single cellular adhesion molecules with nm, pN, and sub-second resolutions. MFM uses novel FRET-based molecular tension sensors that bind to a glass coverslip and present a binding site for integrins, a ubiquitous class of cell adhesion proteins. Cell-generated forces stretch the MFM sensor molecules, resulting in decreased FRET with increasing load that can be imaged at the single-molecule level. Human foreskin fibroblasts adhere to surfaces functionalized with the MFM probes and develop robust focal adhesions. FRET values measured using MFM indicate forces of between 1 and 4 pN per integrin, thus providing the first direct measurement of the tension per integrin molecule necessary to form stable adhesions. The relatively narrow force distribution suggests that mechanical tension is subject to exquisite feedback and control at the molecular level. [Preview Abstract] |
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