Browsing by Subject "Single molecule"
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Item Characterization of HIV-1 Reverse Transcriptase substrate specificity by conformationally sensitive fluorescence(2010-12) Kellinger, Matthew William; Johnson, Kenneth Allen; Dalby, Kevin; Robertus, Jon; Russell, Rick; Zhang, Yan JessieWe have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). The result is a polymerase that is kinetically indistinguishable from the wild-type enzyme, but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding. Using this system, we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding. In doing so, we define the role of induced-fit in nucleotide specificity and mismatch discrimination. Additionally, we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI’s) used to combat HIV infection including 3TC (Lamivudine), FTC (Emtricitabine), and AZT (Zidovudine) for the wild-type polymerase and mutants with clinical resistance to these compounds. Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors. The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change. The apparent Kd (Kd,app) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium. The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association. We show that DNA binding is governed by a two-step process where a fast initial association is followed by a second, slow isomerization that is off the pathway for nucleotide binding and incorporation. Finally, we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase. We find that primer termination with AZT results in DNA that fails to translocate, therefore occluding the next nucleotide from binding. This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP- or pyrophosphate-mediated AZT excision; thereby rescuing productive polymerization. This finding represents the first kinetic measurement of DNA translocation by a polymerase.Item Direct measurement of the energy landscape of ligand-receptor interactions(2010-08) Schwemmer, Frank Heinz, 1986-; Florin, Ernst-Ludwig; Shubeita, George T.In this thesis, a novel single molecule technique will be presented that will, for the first time, give direct access to the interaction energy landscapes of small molecules. The technique relies on the interpretation of thermal position fluctuations of a colloidal probe particle tethered to the molecular complex of interest and a geometrical amplification effect that converts Ångstrom scale fluctuations of the ligand in the binding pocket of the receptor to tens of nanometer fluctuation of the bead. The position of the bead is measured with 0.5 MHz bandwidth and 2 nm spatial resolution. The surface characteristic of the substrate was found to be critical for this new technique and various surface effects were observed. Methods were developed to block nonspecific interaction between the surfaces. The mobility of specifically bound particles was found to depend strongly on the density of specific bonds and the length of the molecular complex; low concentration and short linker lead to slow ligand-receptor mediated surface diffusion, high concentration and/or long linkers to an immobilization of the particle. Transient bond formation was observed for the intermediate range. Details of the interaction energy landscape were not resolved. However, a systematic change in the linker length from 22 Å to 29 Å led to a corresponding change in the lateral position fluctuations from 12.9 nm to 13.2 nm in excellent agreement with our theoretical calculations, confirming the geometrical amplification effect. Also, a new phenomenon of nanometer scale friction in the gap between the bead and the surface was discovered. In summary, the results underline that the novel technique might be able to measure details of the interaction energy landscape of a specific ligand-receptor bond and thus test theoretical predictions for its shape.Item Dynamical simulation of molecular scale systems : methods and applications(2010-12) Lu, Chun-Yaung; Henkelman, Graeme; Rossky, Peter J.; Makarov, Dmitrii E.; Vanden Bout, David A.; Truskett, Thomas M.Rare-event phenomena are ubiquitous in nature. We propose a new strategy, kappa-dynamics, to model rare event dynamics. In this methodology we only assume that the important rare-event dynamics obey first-order kinetics. Exact rates are not required in the calculation and the reaction path is determined on the fly. kappa-dynamics is highly parallelizable and can be implemented on computer clusters and distributed machines. Theoretical derivations and several examples of atomic scale dynamics are presented. With single-molecule (SM) techniques, the individual molecular process can be resolved without being averaged over the ensemble. However, factors such as apparatus stability, background level, and data quality will limit the amount of information being collected. We found that the correlation function calculated from the finite-size SM rotational diffusion trajectory will deviate from its true value. Therefore, care must be taken not to interpret the difference as the evidence of new dynamics occurred in the system. We also proposed an algorithm of single fluorophore orientation reconstruction which converts three measured intensities {I₀,I₄₅,I₉₀} to the dipole orientation. Fluctuations in the detected signals caused by the shot noise result in a different prediction from the true orientation. This difference should not be interpreted as the evidence of the nonisotropic rotational motion. Catalytic reactions are also governed by the rare-events. Studying the dynamics of catalytic processes is an important subject since the more we learn, the more we can improve current catalysts. Fuel cells have become a promising energy source in the past decade. The key to make a high performance cell while keeping the price low is the choice of a suitable catalyst at the electrodes. Density functional theory calculations are carried out to study the role of geometric relaxation in the oxygen-reduction reaction for nanoparticle of various transition metals. Our calculations of Pt nanoparticles show that the structural deformation induced by atomic oxygen binding can energetically stabilize the oxidized states and thus reduces the catalytic activity. The catalytic performance can be improved by making alloys with less deformable metals.Item Interactions of single and few organic molecules with SERS hot spots investigated by orientational imaging and super-resolution optical imaging(2012-05) Stranahan, Sarah Marie; Willets, Katherine A.Dynamics between organic molecules and surface enhanced Raman scattering (SERS) hot spots are extracted from far-field optical images by two experimental methods presented in this thesis: orientational imaging and super-resolution optical imaging. We introduce SERS orientational imaging as an all-optical technique able to determine the three-dimensional orientations of SERS-active Ag nanoparticle dimers. This is accomplished by observing lobe positions in SERS emission patterns formed by the directional polarization of SERS emission along the longitudinal axis of the dimer. We further extend this technique to discriminate nanoparticle dimers from higher order aggregates by observing the wavelength-dependence of SERS emission patterns, which are unchanged in nanoparticle dimers, but show differences in higher order aggregates involving two or more nanoparticle junctions. Dynamic fluctuations in the SERS emission pattern lobes are observed in aggregates labeled with low dye concentrations, as molecules diffuse into regions of higher electromagnetic enhancement in multiple nanoparticle junctions. In order to investigate these dynamic interactions between single organic molecules and nanoparticle hot spots we present the first super-resolution optical images of single-molecule SERS (SM-SERS), introducing super-resolution imaging as a powerful new tool for SM-SERS studies. Mapping the dynamic movement of SM-SERS centroid positions with +/- 5 nm resolution reveals the position-dependent SERS intensity as the centroid samples different positions in space. We have proposed that the diffusion of the SERS centroid is due to diffusion of a single molecule on the surface of the nanoparticle, which leads to changes in coupling between the scattering dipole and the optical near field of the nanoparticle. Finally, we combine an isotope-edited bi-analyte SERS spectral approach with super-resolution optical imaging and atomic force microscopy (AFM) structural analysis for a more complete picture of molecular dynamics in SERS hot spots. We demonstrate the ability to observe multiple molecule dynamics in a single hot spot and show that in addition to the single-molecule regime, a "few" molecule regime is able to report on position-dependent SERS intensities in a hot spot. Furthermore, we are able to identify multiple local hot spots in single nanoparticle aggregates.Item Investigating self-assembly of linked oligomeric PPV-based materials(2014-12) Ingle, Shauna Elizabeth; Vanden Bout, David A.Single molecule wide-field polarization fluorescence imaging is an experimental method to determine the self-assembly of molecules dispersed in a thin film. Through a combination of wide-field imaging and confocal spectroscopy, the effect of synthetic structure of the oligomeric PPV-based materials was investigated to understand the effect of conjugation length, role of hydrogen bonding side chains, and influence of regioregularity on controlling chromophore folding. By studying alkoxy-linked and alkyl-linked bis(2-ethylhexyl)-p-phenylene vinylene (BEH-PPV) units of varying lengths (three, five, or seven), it was determined that conjugation length controlled the extent of molecular ordering and emission properties. Comparison of the experimental results to molecular dynamics simulations performed by collaborators confirmed that the materials became increasingly ordered as conjugation length increased. Further regulation of the assembly can be obtained through inclusion of hydrogen bonding side chains as seen in the altered amine and carboxylic acid alkoxy-linked trimer BEH-PPV in contrast to the bulky side chain tert-butyl trimer. The study of regio-regular (RR) and regio-random (RRa) alkoxy-linked pentamer poly(2-methoxy-5-(2’-ethylhexyloxy)-p-phenylenevinylene) (MEH-PPV) illustrates the limited effect of regioregularity of the side chains on self-assembly. Through synthetic structure, it is possible to design highly ordered materials through control of conjugation length and selection of side chains.