Bulletin of the American Physical Society
APS March Meeting 2010
Volume 55, Number 2
Monday–Friday, March 15–19, 2010; Portland, Oregon
Session D10: Focus Session: Single Molecule Biophysics and Chemical Physics III |
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Sponsoring Units: DCP DBP DPOLY Chair: Rob Phillips, California Institute of Technology Room: A106 |
Monday, March 15, 2010 2:30PM - 3:06PM |
D10.00001: Photon-by-photon trajectories of single protein molecules folding and unfolding Invited Speaker: The transition path time in kinetics is the tiny fraction of an equilibrium trajectory for a single molecule when the transition actually happens and has not been measured for any molecular process in solution. From measurements of photon-by-photon trajectories for fluorophore-labeled single protein molecules undergoing folding and unfolding transitions we have determined that the upper bound for the transition path time is more than 10,000-fold less than the mean first passage time, consistent with a Kramers' analysis of diffusive barrier crossings. [Preview Abstract] |
Monday, March 15, 2010 3:06PM - 3:18PM |
D10.00002: Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers Wesley Wong Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009). [Preview Abstract] |
Monday, March 15, 2010 3:18PM - 3:30PM |
D10.00003: Single Molecule Manipulation and Self-Assembly of Amyloid $\beta $/A4 Precursor Protein on Ag(111) Sajida Khan, K. Clark, A. Deshpande, S.-W. Hla We perform a single molecule level study of the RERMS sequence of amyloid $\beta $/A4 precursor protein fragment on a clean Ag(111) surface using a low-temperature scanning-tunneling-microscope (STM) system at 5K. The mechanical stability of individual protein fragments are checked by laterally manipulating them with the STM tip. Moreover, we are able to form a well ordered two-dimensional layer of the protein fragment by increasing the deposition time. A unit cell has been assigned and a model for the molecular arrangement inside the structure is proposed. This work opens possibilities of using well ordered protein structures on inorganic surfaces for future bimolecular electronic and nano-bio applications. [Preview Abstract] |
Monday, March 15, 2010 3:30PM - 3:42PM |
D10.00004: Single-Molecule Investigations of Organic, Inorganic, and Organometallic Chemical Mechanisms Suzanne Blum Single-molecule fluorescence microscopy has the potential to revolutionize the way in which chemical mechanisms are studied and chemical reactions are improved. Single-molecule techniques will reveal reactivity distributions, which are obscured by traditional ensemble spectroscopic techniques, and will determine active catalysts by direct observation. The potential of single-molecule fluorescence microscopy to address diverse chemical questions, however, has not yet been realized. Previous methods for imaging covalent bond formation were limited to studying chemical reactions that destroyed or produced a fluorophore. We developed a spectator fluorophore imaging technique that does not have this limitation, and which will be generalizable to a large number of chemical reactions, providing unprecedented detail into reaction mechanisms and processes. The formation of individual Pt-S and Pd-P covalent-bonds were imaged on supports similar to those employed in recyclable heterogeneous metal catalysts. A variation in the number of bond-forming events over small surface areas revealed the heterogeneity of the surface's binding properties, which was obscured by traditional ensemble spectroscopy techniques. The application of this technique to the study of broad problems in catalysis will be discussed. [Preview Abstract] |
Monday, March 15, 2010 3:42PM - 4:18PM |
D10.00005: Probing Protein Fluctuations, Folding and Misfolding at Single-molecule Resolution Invited Speaker: The conformational fluctuations and folding of proteins are key for their function in cells and organisms. Conversely, misfolding and aggregation can cause disease, although amyloids with functional significance are also being identified. To better understand these aspects of protein biophysics, we utilize single-molecule fluorescence and complementary methods to directly study complex protein dynamics, structural distributions, and conformational transitions. In one example, we used these methods to investigate disorder and disorder-to-order transitions in intrinsically disordered proteins (IDPs). IDPs are an interesting class of proteins which are relatively unstructured in isolation, but can often fold by interacting with binding partners. These complex systems are increasingly found to play major roles in biology and disease. In one case, we used a combination of single-molecule FRET (smFRET), coincidence and correlation analyses to probe the native structural features of a yeast protein Sup35, whose amyloid state is believed to be used in a beneficial context in yeast. We find that the monomeric protein populates a compact and rapidly fluctuating ensemble of conformations. In another case, we studied the binding-coupled folding of the IDP alpha-synuclein, whose misfolding and aggregation have been linked to Parkinson's disease. Single-molecule measurements directly revealed a complex multi-state folding landscape for this protein. Observations of a transient folding intermediate using microfluidic mixing, and links to misfolding and aggregation will also be discussed. Our results highlight single-molecule methodology that is broadly applicable to map protein folding and misfolding landscapes. [Preview Abstract] |
Monday, March 15, 2010 4:18PM - 4:30PM |
D10.00006: Single Molecule Studies of Anthradithiophene Derivatives W.E.B. Shepherd, A.D. Platt, G. Banton, M.A. Loth, J. Anthony, O. Ostroverkhova Several solution-processable anthradithiophene (ADT) derivatives have been developed which exhibit charge carrier mobilities of $>1 \frac{cm^2}{Vs}$ in films. To better understand charge transport and energy transfer processes in these materials at the molecular level, we apply single-molecule fluorescence spectroscopy (SMFS) techniques to probe effects of intermolecular interactions and external parameters on the molecular properties. In particular, we demonstrate that ADT molecules exhibit high enough quantum yields and photostability to be imaged on a single-molecule level at room temperature. Moreover, we show that the behavior of individual ADT molecules depends on the host matrix (poly (methyl methacrylate) vs a crystalline environment) and is comparable to that of the best fluorophores utilized in SMFS. Finally, we analyze performance of individual ADT molecules depending on their local nanoenvironment (which includes arrangement of the surrounding host molecules and the presence of other guest molecules in the vicinity of the molecule under study). [Preview Abstract] |
Monday, March 15, 2010 4:30PM - 4:42PM |
D10.00007: Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals Shiwei Wu, Gang Han, Delia Milliron, Shaul Aloni, Virginia Altoe, Dmitri Talapin, Bruce Cohen, Jim Schuck The development of probes for single-molecule imaging has dramatically facilitated the study of individual molecules in cells and other complex environments. Single-molecule probes ideally exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-molecule probes are imperfect in several of these aspects, and none have been shown to possess all of these characteristics. In this talk, I will show that individual lanthanide-doped upconverting nanoparticles (UCNPs) emit non-blinking and photostable near-infrared to visible upconverted luminescence when excited by a 980-nm continuous wave laser, suggesting that UCNPs are ideally suited for single-molecule imaging experiments. [Preview Abstract] |
Monday, March 15, 2010 4:42PM - 4:54PM |
D10.00008: Visualization of the mechanochemical coupling in myosin V using deac-aminoATP Takeshi Sakaoto, Martin Webb, Eva Forgacs, Howard White, Jim Sellers MyosinV is a twoheaded motor, which moves processively along an actin with ADP-release as the rate limiting step. The kinetic cycles of the two heads are gated by the internal strain each places on the other Mechanical studies suggest that there is tight coupling (i.e., one ATP is hydrolyzed per power stroke). We investigated the coordination between the ATPase mechanism of the two heads-myosin~Va and directly visualized the binding and dissociation of nucleotide molecules, while simultaneously observing the stepping motion of the myosin V as it moved along an actin filament. To do this, we used an fluorescent labeled ATP analog, deac-aminoATP, which shows a 20fold increase in fluorescent intensity when bound to the active site of myosinV I directly demonstrate tight coupling between myosin V movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision. Supported by K99/R00 NIH grant. [Preview Abstract] |
Monday, March 15, 2010 4:54PM - 5:06PM |
D10.00009: Myosin VI as a transporter and an anchor: A model for kinetics of the motor under load Peiying Chuan, James Spudich, Alexander Dunn Myosin VI is an actin-based motor that is thought to function both as a transporter and an anchor \textit{in vivo}. In an earlier study (Altman et al, \textit{Cell} 2004), inhibition of myosin VI stepping kinetics by load applied using an optical trap was observed at saturating ATP and low ADP concentrations ($<$ 2.5 $\mu $M). A simple mechanism whereby the rate of ADP binding increases exponentially with load was proposed. This model predicts that myosin VI functions primarily as an anchor at loads greater than $\sim $0.5 pN under physiological nucleotide conditions, which is potentially inconsistent with its roles \textit{in vivo}. Here we present myosin VI stepping data taken at a variety of applied loads and ADP concentrations, and show that the Altman model only holds at low ADP concentrations. At higher, physiologically relevant ADP concentrations under load we observe dwell times that are an order of magnitude smaller than predicted by the Altman model. We present a modified model in which applied load alters the equilibrium between two myosin VI states with different nucleotide affinities. This new kinetic scheme accurately describes myosin VI behavior at various nucleotide conditions under a large range of loads, and explains how the motor is able to carry out its roles \textit{in vivo}, both as a force-generating transporter and as an anchor.~~ [Preview Abstract] |
Monday, March 15, 2010 5:06PM - 5:18PM |
D10.00010: Torque generation mechanism of ATP synthase John Miller, Sladjana Maric, M. Scoppa, M. Cheung ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed. [Preview Abstract] |
Monday, March 15, 2010 5:18PM - 5:30PM |
D10.00011: Tracking a Molecular Motor with a Nanoscale Optical Encoder Everett A. Lipman, Charles E. Wickersham, Daniel H. Kerr Optical encoders are commonly used in macroscopic machines, such as desktop printers and astronomical telescopes, to make precise measurements of distance and velocity by translating motion into a periodic signal. We have designed and synthesized self-assembling DNA segments incorporating F\"orster resonance energy transfer acceptor dyes at regular intervals. When one of these ``FRET encoders'' is unwound by a donor-labeled helicase molecule, a periodic fluorescence signal is produced, enabling us to monitor translation and rotation of the helicase. I will describe our methods for synthesizing FRET encoders, and show data indicating constant linear motion of a single \emph{E.~coli} DnaB helicase over a distance of hundreds of base pairs. [Preview Abstract] |
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