Physics
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Item LINK BETWEEN DYNAMICS AND FUNCTION IN SINGLE AND MULTI-SUBUNIT ENZYMES(2010) Chen, Jie; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biopolymers, such as proteins and DNA, are polymers whose three-dimensional conformations dene their biological functions. Current emphasis on structures has greatly advanced our understanding of the functions of biopolymers. However, there is a need to understand the deeper link between biopolymer dynamics and function, because in water and under cellular conditions, everything that biopolymers do can be understood in terms of "the jigglings and wigglings of atoms". These motions arise from thermal noise in the solvent and due to intrinsic motion of the enzymes. In biological systems, the motions are often highly regulated to ensure that cellular processes are executed over the required time scales. For enzymes, which are essentially proteins that catalyze chemical reactions or generate mechanical work, conformational fuctuations are coupled at various stages through interactions with ligands during the catalytic cycle. We have studied two dierent enzymes, dihydrofolate reductase (DHFR), which catalyzes reduction of dihydrofolate to tetrahydrofolate, and RNA polymerase (RNAP from bacteria and Pol II from yeast), which is responsible for RNA synthesis using DNA as a template. In order to study the link between dynamics and function we have developed new methods and extended a variety of computational techniques. For DHFR, we use both evolutionary imprints (SCA) and structure-based perturbation method (SPM) to extract a network of residues that facilitate the transitions between two distinct conformational states (closed and occluded states). The transition kinetics and pathways connecting the closed and occluded states are described using Brownian dynamics (BD) simulation. We found the sliding motion of Met20 loop across helix 2 is involved in the forward and reverse transitions between the closed and occluded states. We also found that cross-linking M16-G121 inhibits both the forward and the reverse transitions. In addition, we showed the transition states of these transitions are broad and resemble high energy states. For RNAP, we focus on the conformational changes of RNAP and DNA in promoter melting process. Using BD, we show that DNA conformation changes in promoter melting occur in three steps. We also show that internal dynamics of RNAP is relevant to facilitate the bending of DNA. For Pol II, the structural transitions between two initiation conformational states and between initiation state and elongation state are studied using SPM and BD. We determine the structural units that regulate structural transitions and describe the transition kinetics. The combination of three dierent methods, SCA, SPM and BD, provide results that are in accord with many experiments. Moreover, our description of the detailed structural transitions in these enzymes lead to new insights and testable predictions in these extraordinarily important enzyme functions.Item TYPE I COLLAGEN HOMOTRIMERS; THEIR ROLE IN COLLAGEN FIBRIL FORMATION AND TISSUE REMODELING(2009) Han, Sejin; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Formation and remodeling of type I collagen fibril networks are paradigms of biopolymer self-assembly, yet many of their aspects remain poorly understood. Type I collagen is the most abundant vertebrate protein which self assembles into fibrils and hierarchical fibril network structures, forming scaffolds of bone, skin, tendons and other tissues. The normal isoform of type I collagen is a heterotrimer of two &alpha1(I) and one &alpha2(I) chains, but homotrimers of three &alpha1(I) chains have been reported, e.g., in cancer and fibrosis. Despite their importance in various disorders, very little is known about potential effects of the type I collagen homotrimers on self-assembly, physical properties, and remodeling of collagen fibrils and fibril networks. Thus, we selected characterization of these effects and understanding the underlying physical mechanisms as the topic of the present thesis. Some of our most important findings were: (i) different nucleation mechanism and morphology in homotrimer fibrils compared to the normal heterotrimers fibrils; (ii) segregation of the homo- and heterotrimers within fibrils; (iii) increased bending rigidity of homotrimer fibrils; and (iv) homotrimer resistance to cleavage by enzymes responsible for fibril degradation and remodeling due to increased triple helix stability at the cleavage site. The corresponding in vitro experiments and theoretical analysis of the results suggested drastically different physics of the fibril networks composed of the homo/heterotrimer mixtures and pointed to a potential role of these physics in various disorders, e.g., in cancer and fibrosis pathology.Item Applications of Multiphoton Imaging Techniques To The Study Of Protein Interactions(2009) Rosales, Tilman J.; Walker, Robert A; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several recent improvements in microscopy have been driven by advances in ultrafast laser technology. The goal of the research described in this dissertation was to develop noninvasive, optically based methods to measure the mobility of macromolecules in biologically relevant systems. These methods exploit advances in ultrafast laser science and recent developments in multiphoton spectroscopy techniques. Each of the techniques described in this dissertation is validated and standardized using well characterized systems. We have explored the following techniques: First, 2-photon 2-color Fluorescence Cross Correlation Spectroscopy (FCCS) a powerful technique to measure dilute protein interactions in living cells. We have used FCCS to probe AR-Tif2 (Androgen Receptor - activating cofactor) interactions in the presence of casodex, an antagonist yielding decreased binding affinity. On a much faster timescale, exploring rotational rather than translational diffusion, we used molecular dynamics simulations of the model probe perylene to show that there is `room to wiggle' (sub-ps libration) within pure hexadecane. Third, combining picosecond and microsecond scales, we built a system to measure both rotational and translational motion in one experiment, using advanced Time Correlated Single Photon Counting (TCSPC) techniques. We have tested our ability to measure and link simultaneously the translational rates and decay rates of Alexa488 dye and other biologically relevant fluorophores. Next, exploiting non-linear vibrational spectroscopy, we have imaged the non-fluorescent molecule NAD+ in DPPC vesicles, the C-H stretch of lipids in vesicles and polystyrene beads, and the O-H stretch of water inside living cells (vs. O-D) to demonstrate the chemically selective imaging capabilities of Coherent Anti-Stokes Raman Spectroscopy (CARS) Microscopy. Most recently, we have built a STED (STimulated Emission Depletion) Microscope capable of extracting fluorescent images well below the diffraction limit. The STED microscope was tested using both 170nm fluorescent beads and a novel photochromic dye.Item Dendritic Integration and Reciprocal Inhibition in the Retina(2008-11-17) Grimes, William Norman; Walker, Robert; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mammalian retina is capable of signaling over a vast range of mean light levels (~10^10). Such a large dynamic range is achieved by segregating signals into contrasting pathways and utilizing excitatory and inhibitory neural circuits. The goal of this study was to elucidate subcellular mechanisms responsible for shaping dendritic computation and reciprocal inhibition within the retinal circuitry. Amacrine cells make up a unique class of inhibitory interneurons which lack anatomically distinct input and output structures. Although these interneurons clearly play important roles in complex visual processing, there is relatively little known about the ~30 subtypes. A17 amacrine cells have been shown to shape the time course of visual signaling in vivo. Intuition might suggest that a wide field (~400 µm) interneuron, such as A17, would provide long range lateral inhibition or center surround inhibition. However, using multi-disciplinary approaches, we have uncovered multiple mechanisms which underlie dendritic integration and synaptic transmission in A17 that allow it to respond with a high degree of synapse specificity. Additionally, these mechanisms work in concert with post-synaptic mechanisms to extend the dynamic range of reciprocal inhibition in the inner retina.Item Protein folding and amyloid formation in various environments(2008-11-21) O'Brien, Edward Patrick; Thirumalai, Devarajan; Brooks, Bernard; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding and predicting the effect of various environments that differ in terms of pH and the presence of cosolutes and macromolecules on protein properties is a formidable challenge. Yet this knowledge is crucial in understanding the effect of cellular environments on a protein. By combining thermodynamic theories of solution condition effects with statistical mechanics and computer simulations we develop a molecular perspective of protein folding and amyloid formation that was previously unobtainable. The resulting Molecular Transfer Model offers, in some instances, quantitatively accurate predictions of cosolute and pH effects on various protein properties. We show that protein denatured state properties can change significantly with osmolyte concentration, and that residual structure can persist at high denaturant concentrations. We study the single molecule mechanical unfolding of proteins at various pH values and varying osmolyte and denaturant concentrations. We find that the the effect of varying solution conditions on a protein under tension can be understood and qualitatively predicted based on knowledge of that protein's behavior in the absence of force. We test the accuracy of FRET inferred denatured state properties and find that currently, only qualitative estimates of denatured state properties can be obtained with these experimental methods. We also explore the factors governing helix formation in peptides confined to carbon nanotubes. We find that the interplay of the peptide's sequence and dimensions, the nanotube's diameter, hydrophobicity and chemical heterogeneity, lead to a rich diversity of behavior in helix formation. We determine the structural and thermodynamic basis for the dock-lock mechanism of peptide deposition to a mature amyloid fibril. We find multiple basins of attraction on the free energy surface associated with structural transitions of the adding monomer. The models we introduce offer a better understanding of protein folding and amyloid formation in various environments and take us closer to understanding and predicting how the complex environment of the cell can effect protein properties.Item Polymer Concepts in Biophysics(2008-04-26) Morrison, Gregory Charles; Thirumalai, Devarajan; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recent advent of experimental techniques that study biological systems on the level of a single molecule have lead to a number of exciting new results. These experiments have a variety of applications in understanding both the kinetics and equilibrium properties of biomolecules. By applying the concepts of polymer physics to these single molecule experiments, we are able to more fully understand the physical picture underlying a number of experimental observations. In this thesis, we use a variety of polymer models to develop a better understanding of many single molecule experiments. We show that the kinetics of loop formation in biopolymers can be generally understood as a combination of an equilibrium and dynamic part for a number of different polymer models. We study the extension of a homopolymer as a function of applied tension, and develop a simple theoretical framework for determining the effect of interactions on the stretching of the chain. We show that the measured hopping rates in a laser optical tweezer experiment are necessarily altered by the experimental setup, and suggest a method to accurately infer the correct hopping rates using accurately measured free energy profiles. We show that the effect of the experimental setup can be understood using a novel polymer model. Finally, we propose a Hamiltonian-based method to study the properties of spherically confined wormlike chains, which accurately determines the equilibrium properties of the system for strongly confined chains. In these studies, we are able to better understand the behavior of many disparate systems using relatively simple arguments from polymer theory.Item A Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modeling(2007-04-27) Ferguson, Matthew Lee; Nossal, Ralph J; Losert, Woflgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triske- lion) that assembles into polyhedral baskets principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends on the pH, with baskets forming more readily at low pH and less readily at high pH. We have developed procedures, based on static and dynamic light scattering, to determine the radius of gyration, Rg, and hydrodynamic radius, RH, of isolated triskelia under conditions where basket assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending of the legs and a puckering at the vertex. We also show that the values of Rg and RH measured for clathrin triskelia in solution are qualitatively consistent with the conformation of an individual triskelion that is part of a "D6 barrel" basket assembly measured by cryo-EM tomography. We extended this study by performing small angle neutron scattering (SANS) experiments on isolated triskelia in solution under conditions where baskets do not assemble. SANS experiments were consistent with previous static light scattering ex- periments but showed a shoulder in the scattering function at intermediate q-values just beyond the central diffraction peak (the Guinier regime). Theoretical calcula- tions based on rigid bead models of a triskelion showed well-defined features in this region different from the experiment. A flexible bead-spring model of a triskelion and Brownian dynamics simulations were used to generate a time averaged scattering function. This model adequately described the experimental data for flexibilities close to previous estimates from the analysis of electron micrographs.Item Stretching Biomolecules(2005-11-23) Hyeon, Changbong; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biomolecular self-assembly is the complicated processes characterized by broad and hierarchical structure of energy, length, and time scales. Various experimental tools have, for decades, been used to understand the principles governing the dynamics of biomolecular systems. The recent advent of single molecule force experiments has expanded our perspective on the energetics of biomolecules, explicitly showing the diverse traces of the individual molecules undergoing heterogeneous processes. Based on theoretical arguments and Langevin dynamics simulations of coarse-grained models, I suggest that the following aspects of biomolecules can be elucidated through the force experiments. (1) The energy landscape roughness can be directly measured if the force experiment is performed in varying temperature. (2) The diverse nature of biomolecular energetics, reflecting the underlying complexity of energy landscape, manifests itself when the molecule is subject to various conditions. As a simple example, thermodynamic and kinetic properties of two-state folding RNA hairpins are investigated at varying temperature and force. We show the thermal and the force induced unfolding/refolding dynamics are vastly different. (3) The free energy landscape of a molecule deforms its terrain differently depending on the nature of external control variable. Force plays a different role from temperature when it is exerted on the molecule. The explicit computations and comparisons of free energy profiles along the reaction coordinate find a Hammond behavior in force but not in temperature. (4) Native topology and polymeric nature of biomolecule determines the force-induced unfolding pathway. (5) There exist pulling speed-dependent unfolding pathways in biomolecules if the molecule consists of multiple subdomains. When combined with theory, single molecule force experiments can unravel the rich nature of biomolecular free energy landscape. All of the theoretical predictions made in the present work are amenable to the future experiments.Item Distinguishing Modes of Eukaryotic Gradient Sensing(2005-08-25) Skupsky, Ron; Losert, Wolfgang; Nossal, Ralph J; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The behaviors of biological systems depend on complex networks of interactions between large numbers of components. The network of interactions that allows biological cells to detect and respond to external gradients of small molecules with directed movement is an example where many of the relevant components have been identified. This behavior, called chemotaxis, is essential for biological functions ranging from immune response in higher animals to the food gathering and social behavior of ameboid cells. Gradient sensing is the component of this behavior whereby cells transduce the spatio-temporal information in the external stimulus into an internal distribution of molecules that mediate the mechanical and morphological changes necessary for movement. Signaling by membrane lipids, in particular 3' phosphoinositides (3'PIs), is thought to play an important role in this transduction. Key features of the network of interactions that regulates the dynamics of these lipids are coupled positive feedbacks that might lead to response bifurcations and the involvement of molecules that translocate from the cytosol to the membrane, coupling responses at distant point on the cell surface. Both are likely to play important roles in amplifying cellular responses and shaping their qualitative features. To better understand the network of interactions that regulates 3'PI dynamics in gradient sensing, we develop a computational model at an intermediate level of detail. To investigate how the qualitative features of cellular response depend on the structure of this network, we define four variants of our model by adjusting the effectiveness of the included feedback loops and the importance of translocating molecules in response amplification. Simulations of characteristic responses suggest that differences between our model variants are most evident at transitions between efficient gradient detection and failure. Based on these results, we propose criteria to distinguish between possible modes of gradient sensing in real cells, where many biochemical parameters may be unknown. We also identify constraints on parameters required for efficient gradient detection. Finally, our analysis suggests how a cell might transition between responsiveness and non-responsiveness, and between different modes of gradient sensing, by adjusting its biochemical parameters.