UMD Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/3

New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.

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THERMODYNAMIC PROPERTIES OF THE UNFOLDED ENSEMBLE OF PROTEINS
(2010) DESAI, TANAY MAHESH; MUNOZ, VICTOR; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
A random coil, whose size is determined by its excluded volume, and net energetic interactions with its environment, has served as a representation of the unfolded ensemble of proteins. The work in this thesis involves equilibrium, nuclear magnetic resonance and time-resolved kinetics spectroscopic studies on the unfolded ensemble of BBL, a globally downhill folding 40-residue protein involved the Krebs cycle of E. coli, in its acid-denatured state, and on a sequence-randomized version of this protein. The effect of variability in thermodynamic conditions, such as temperature and the presence of added chaotropes or kosmotropes, on the equilibrium properties and reconfiguration dynamics of the unfolded state, have been deduced in the absence of competition with folding events at low pH. The unfolded ensemble experiences expansion and collapse to varying degrees in response to changes in these conditions. Individual interactions of residues of the protein with the solvent and the cosolvent (direct interactions), and the properties of the solution itself (indirect interactions) are together critical to the unfolded chain's properties and have been quantitatively estimated. Unfolded, protonated BBL can be refolded by tuning the properties of the solvent by addition of kosmotropic salts. Electrostatic interactions turn out to be essential for folding cooperativity, while solvent-mediated changes in the hydrophobic effect can promote structure formation but cannot induce long-range thermodynamic connectivity in the protein. The effect of sequence on the properties of heteropolymers is also tested with a randomized version of BBL's sequence. Chain radii of gyration, and the degree and rate of hydrophobic collapse depend on the composition of the sequence, viz. hydrophilic versus hydrophobic content. However, the ability to maximize stabilizing interactions and adopt compact conformations is more evident in naturally selected protein sequences versus designed heteropolymers. Chain reconfiguration of unfolded BBL takes place in ∼1/(100 ns), in agreement with theoretical estimates of homopolymer collapse rates. The refolding dynamics of salt-refolded BBL in the range of 1/(6 μs) at 320 K, emerge as being independent of the degree of folding or protonation of the chain, a result in keeping with the description of dynamics in BBL as oscillations in a single, smooth harmonic potential well, which only varies in its position and curvature with varying thermodynamic conditions.
A biophysical evaluation of cell-substrate interactions during spreading, migration and neuron differentiation
(2010) Norman, Leann Lynn; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The development of engineered scaffolds has become a popular current avenue to treat numerous traumas and disease. In order to optimize the efficiency of these treatments, it is necessary to have a more thorough understanding of how cells interact with their substrate and how these interactions directly affect cellular behavior. Cell spreading is a critical component of numerous biological phenomena, including embryonic development, cancer metastasis, immune response, and wound healing. Along with spreading, cell adhesion and migration are all strongly dependent on the interactions between the cell and its substrate. Cell-substrate interactions can affect critical cellular mechanisms including internal cellular signaling, protein synthesis, differentiation, and replication and also influence the magnitude of adherence and motility. In an effort to better understand cell-substrate interactions we characterize the initial stages of cell spreading and blebbing using cell-substrate specific microscopy techniques, and identify the effects of cytoskeletal disruption and membrane modification on surface interactions and spreading. We identify that blebs appear after a sharp change in cellular tension, such as following rapid cell-substrate detachment with trypsin. An increased lag phase of spreading appears with increased blebbing; however, blebbing can be tuned by supplying the cell with more time to perform plasma membrane recycling. We developed software algorithms to detect individual bleb dynamics from TIRF and IRM images, and characterize three types of bleb-adhesion behaviors. Overall, we show that blebs initially create the first adhesion sites for the cell during spreading; however, their continuous protrusion and retraction events contribute to the slow spreading period prior to fast growth. In addition, we identify the elastic modulus of the rat cortex and characterize a polyacrylamide gel system that evaluates the effects of substrate stiffness on cortical outgrowth. Remarkably, we illustrate that cortical neuron differentiation and outgrowth are insensitive to substrate stiffness, and observe only morphological differences between laminin versus PDL-coated substrates. Together, this research identifies cell-specific behaviors critical to spreading and migration. The thorough evaluations of spreading and migration behavior presented here contribute to the understanding of critical cellular phenomena and suggest potential therapeutic targets for treatment of cardiovascular disease and neurological disorders.
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.
Fluorescence and NMR Studies of the Role of Metal Ions in HIV-1 Genomic RNA Dimerization and Maturation
(2009) Lee, Hui-Wen; Fushman, David; Marino, John P.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The dimerization initiation site (DIS) is an essential RNA element responsible for dimerization of HIV-1 genomic RNA through a kissing loop interaction. The DIS loop contains six auto-complementary nucleotides stabilized by 5'- and 3'-flanking purines. NCp7 chaperone protein catalyzes conversion of an intermediate DIS kissing dimer to a more thermodynamically stable extended duplex dimer in the presence of Mg2+. Sequence constructs intended to model the extended duplex, (DIS 21), and the kissing dimer, DIS23(GA)*DIS23(HxUC), were designed to examine the structural information and biochemical behaviors during maturation. We introduced the fluorescent labeling, 2-aminopurine (2-AP) into these RNA constructs, to finely probe structural transition and local dynamics accompanied by the formation of the DIS dimer. The 2-AP nucleotides were inserted either in the DIS loop or junction to study loop-loop interaction or purine base stacking conformation at the junction responding to the metal ion effect. High resolution NMR methods were then used to probe structural changes associated with mono versus divalent cation binding to the DIS dimers and also determine the Mg2+ binding sites. Significant chemical shift perturbations (CSP) were found upon Mg2+ binding and used to map structural changes. Further Mn2+ paramagnetic relaxation enhancement (PRE) experiments provided evidence for specific Mg2+ ion binding are localized around the 5' purine bases in both the extended duplex and kissing dimers with profound line broadening effects. Mapping the CSP and PRE data onto the available X-ray crystal and NMR solution structures allowed localization of specific Mg2+ ions at binding sites on the DIS dimers created by the unpaired flanking DIS loop purine nucleotides. Our data indicates that the conformations that are metal cation dependent. These findings are consistent with previous results that suggested a role for divalent metal cations in stabilizing the DIS kissing dimer structure and influencing its maturation to an extended duplex form through interactions with the DIS loop.
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.
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.
STRUCTURE FUNCTION DIVERSITY WITHIN THE PHOSPHOENOLPYRUVATE MUTASE / ISOCITRATE LYASE SUPERFAMILY AS REVEALED BY THE ENZYMES OXALOACETATE DECARBOXYLASE AND 2,3-DIMETHYLMALATE LYASE
(2008) Narayanan, Buvaneswari Coimbatore; Herzberg, Osnat; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Two members of the phosphoenolpyruvate mutase (PEPM) / isocitrate lyase (ICL) superfamily were investigated to study their structure-function relationships and to identify sequence signatures that define a particular function. The first enzyme (PA4872) was a protein of unknown function from Pseudomonas aeruginosa. The second enzyme from Aspergillus niger (An07g08390) was thought to be an oxaloacetate acetyl hydrolase (OAH) because of its high sequence identity (~60%) to an enzyme with confirmed OAH activity. The X-ray crystal structure determination of PA4872 revealed unique features that guided the design of biochemical experiments, which ultimately led to the discovery that the enzyme is an oxaloacetate decarboxylase (OAD). Two structures of An07g08390, one with bound Mg2+ and the second with bound Mn2+ and the inhibitor 3,3-difluorooxaloacetate, were determined. The functional studies demonstrated that although the enzyme has OAH activity, it has a far better activity as a 2R,3S-dimethylmalate lyase (DMML). The active site structure of DMML indicated a proline residue (Pro240) as a marker of DMML function along with confirming the conserved locations of previously established signature residues for lyase activity. OAD is the founding member of a family within the PEPM / ICL superfamily and thus defines the function of the remaining family members. However, the biological context in which OAD functions remains unknown. DMML is known to function in the nicotinate catabolism pathway but not all the members of the pathway are present in A. niger. Transcriptome analysis suggests that the DMML encoding gene is under carbon catabolite repression but the pathway in which the enzyme functions has not yet been identified.
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.
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.
Energetics of Drug Interactions
(2008-11-26) Todorova, Niya Ancheva; Kelman, Zvi; Schwarz, Frederick P.; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The goal of our research is to determine in terms of thermodynamic change of state functions the effects of experimental factors, such as water, mutagenesis, or the presence of a second substrate on the energetics of drug-inhibitor binding interactions. The binding of non-steroidal anti-inflammatory drugs within the rigid cavities of cyclodextrins was investigated by titration calorimetry and spectrofluorimetry. Loss of bulk water structure upon drug binding in the smaller hydrophobic β-cyclodextrin cavity results in an increase in the binding entropy, while restriction of the configurations of the drug in the cavity decreases the binding entropy. This restriction in the hydrophobic β-cyclodextrin cavity enhances the binding enthalpies so that the β-cyclodextrin binding reactions are enthalpy-driven. In the larger γ-cyclodextrin cavity, water is retained so that, not only are the interactions between the drug and the cavity reduced, there is an increase in the drug configurations resulting in increases in the binding entropies and the binding reactions become entropically-driven. These binding reactions also manifest enthalpy-entropy compensation where changes in the binding enthalpies are compensated by changes in the binding entropies. In drug binding to the more flexible p38α MAP kinase mutants, a single-point C→S mutation distal from the binding site, changes the interaction between the N- and C-terminal structural domains of the kinase as evident in differential scanning calorimetry. Calorimetric results show that drug-inhibitor binding affinities to kinase increase with size of the drugs since the binding reactions are all enthalpically-driven. Drug-inhibitors binding to trimeric human purine nucleoside phosphorylase were investigated by calorimetry in the presence of its second substrate, inorganic phosphate (Pi). Increasing concentrations of Pi modulates the driving-nature of the binding reaction, so that the acyclovir binding almost exclusively to the purine substrate binding site becomes more entropically-driven, while the binding reactions of ganciclovir and 9-benzylguanine interacting also with the adjacent Pi substrate site become more enthalpically-driven. A novel calorimetric enzyme activity assay at the low dissociation concentrations of the phosphorylase show an increase in the enzyme activity at low Pi concentrations, but also a decrease in the 9-benzylguanine binding affinity since this drug also interacts with an adjacent subunit.

UMD Theses and Dissertations

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