Browsing by Subject "Biophysics"
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Item Biophysical characterization of several hemoproteins from the photosynthetic bacteria, Chromatium vinosum and Rhodospirillum rubrum(Texas Tech University, 1985-08) Gaul, Dale FrancisNot availableItem Development of accurate and efficient models for biological molecules(2011-12) Wu, Johnny Chung; Ren, PengyuThe abnormal expression or function of biological molecules, such as nucleic acids, proteins, or other small organic molecules, lead to the majority of diseases. Consequently, understanding the structure and function of these molecules through modeling can provide insight and perhaps suggest treatment for diseases. However, biologically relevant molecular phenomenon can vary vastly in the nature of their interactions and different classes of models are required to accommodate for this diversity. The objective of this thesis is to develop models for small molecules, amino acid peptides, and nucleic acids. A physical polarizable molecular mechanics model is described to accurately represent small molecules and single atom ions and applied to predict experimentally measurable thermodynamic properties such as hydration and binding free energies. A novel physical coarse-grain model based on Gay-Berne potentials and electrostatic multipoles has been developed for short peptides. The fraction of residues that adopt the alpha-helix conformation agrees with all-atom molecule dynamics results. Finally, a statistically-derived model based on sequence comparative sequence alignments is developed and applied to improve folding accuracy of RNA molecules.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 Dissecting the relationship between protein structure and sequence evolution(2015-05) Shahmoradi, Amir; Mahajan, Swadesh M.; Wilke, C. (Claus); Orbach, Raymond L; Marder, Michael P; Gordon, Vernita D; Press, William HWhat can protein structure tell us about protein evolutionary dynamics? Despite extensive variety in their native structures, from hyper-thermostable to intrinsically disordered, all proteins share a common feature: flexibility and dynamics at different levels of structure. In addition to spatial dynamics, proteins are also highly evolutionary dynamic polymers, exhibiting variability in their amino acid sequences on evolutionary timescales. Significant variations can be observed in the amino acid sequences of the divergent members of a single protein family, while their native conformations and biological functions remain almost conserved among all members of the family. These evolutionary variations can be due to a combination of point mutations, insertions, deletions or sometimes the rearrangement of domains in the protein sequence. In recent years, it has become increasingly evident that the dynamics of proteins in space and time domains -- corresponding to structural and evolutionary variations -- mutually influence each other at the amino acid level. In particular, it is generally observed that the amino acids in the core of protein are more conserved than the amino acids on the surface. Some site-specific structural quantities have been already identified that are capable of explaining the general patterns of sequence variability in globular proteins. A prominent example is the amino acid exposure to solvent molecules -- typically water -- which surround proteins in vivo. Furthermore, some partial associations between the local flexibility, packing density and sequence variability can be also observed among globular proteins. There is however no consensus as to which set of structural characteristics play the dominant role in sequence evolution. The strength of sequence--structure correlations also appear to vary widely from one protein to another, with Spearman's correlation strength ρ ∈ [0.1,0.8]. Throughout a series of works summarized in the following chapters, first I explore the wide spectrum of structural determinants of sequence evolution, their interrelationships, and their role in the evolutionary dynamics of protein. I find that amino acid sites that are important for the overall stability of protein structure in general tend to be highly conserved. In other words, any amino acid substitution that results in a significant change of the potential energy landscape and thus the native conformation of protein, is disruptive and hence occurs less frequently on evolutionary timescale. I also find that long-range interactions among individual amino acids play a weak but non-negligible role in site-specific evolution of proteins and their inclusion generally results in better predictions of sequence evolution from protein structure. Then, I present the results from a comprehensive search for the potential biophysical and structural determinants of protein evolution by studying >200 structural and evolutionary characteristics of proteins in a dataset of viral and enzymatic proteins. I discuss the main protein properties responsible for the general patterns of protein evolution, and identify sequence divergence as the main determinant of the strengths of virtually all structure-evolution relationships, explaining ~ 10 - 30% of the observed variation in sequence-structure relations. In addition to sequence divergence, I identify several protein structural properties that are moderately but significantly coupled with the strength of sequence-structure relations. In particular, proteins with more homogeneous back-bone hydrogen bond energies, corresponding to proteins containing large fractions of helical secondary structures and low fraction of beta sheets tend to have the strongest sequence-structure relations.Item Expression and function of the JunD transcription Factors: JunD-FL and Delta-JunD(Texas Tech University, 2003-08) Short, John DJunD is a member of the Jun family of basic region leucine zipper proteins that can form homodimers or form heterodimers with other Jun family members (c-Jun and JunB) or Fos family members (c-Fos, FosB, Fra-1, Fra-2). Collectively, these dimer combinations make up the Activator Protein-1 (AP-1) transcription factor. AP-1 binds to the TPA-response element, TGAG/CTCA, within the promoter of a wide-range of genes, many of which are important for cell growth regulation. JunD functions as a negative regulator of transformed cell growth and antagonizes transformation by the ras oncogene. JunD also functions to protect cells from premature senescence and apoptosis. The junD gene, like the other jun genes, is intronless and generates a single mRNA. However, we have found that the JunD mRNA generates two predominant JunD protein isoforms through alternative translational initiation. The larger JunD isoform, JunD-FL (39 kD) is generated by translational initiation at the first AUG downstream of the m7-G cap, and the smaller JunD isoform, AJunD (34 kD), is generated by translational initiation at the third AUG codon from the m7-G cap. These AUG codons are in-frame, making JunD-FL a 48-amino acid N-terminal extension of ÄJunD. We have also identified four other potential translational initiation events that occur at both AUG and non-AUG start codons within the JunD mRNA, suggesting that there are six peptides generated from the JunD mRNA. JunD-FL and AJunD function differentially within the cell. Both JunD isoforms bind to Jun-N-terminal kinases (JNKs), but JNKs more potently stimulate transactivation of JunD-FL in vitro. In addition, JunD-FL interacts with the Menin tumor suppressor protein. Menin inhibits transcriptional activity of JunD-FL in vitro, but does not bind to or inhibit ÄJunD transcriptional activity. We have identified several putative target genes of JunD-FL and ÄJunD, including the nuclear orphan receptor nur77, which is involved in cell growth arrest and cellular apoptosis. We found that Nur77 is positively regulated by JunD-FL and ÄJunD in a similar manner but is negatively regulated by c-Jun.Item Optimization of force fields for molecular dynamics(2014-12) Di Pierro, Michele; Elber, RonA technology for optimization of potential parameters from condensed phase simulations (POP) is discussed and illustrated. It is based on direct calculations of the derivatives of macroscopic observables with respect to the potential parameters. The derivatives are used in a local minimization scheme, comparing simulated and experimental data. In particular, we show that the Newton Trust-Region protocol allows for accurate and robust optimization. POP is illustrated for a toy problem of alanine dipeptide and is applied to folding of the peptide WAAAH. The helix fraction is highly sensitive to the potential parameters while the slope of the melting curve is not. The sensitivity variations make it difficult to satisfy both observations simultaneously. We conjecture that there is no set of parameters that reproduces experimental melting curves of short peptides that are modeled with the usual functional form of a force field. We then apply the newly developed technology to study the liquid mixture of tert-butanol and water. We are able to obtain, after 4 iterations, the correct phase behavior and accurately predict the value of the Kirkwood Buff (KB) integrals. We further illustrate that a potential that is determined solely by KB information, or the pair correlation function, is not necessarily unique.Item Submicroscopic characterization of biopolymer networks in solution by Thermal Noise Imaging(2013-05) Bartsch, Tobias Fabian; Florin, Ernst-Ludwig; Shubeita, George T; Aldrich, Richard W; Demkov, Alex A; Fink, ManfredBiopolymer networks display a wide range of interesting mechanical properties that are essential for living organisms. For example, a highly nonlinear elastic response to strain gives biopolymer networks the ability to comply with small stresses but to resist large ones. These macroscopic mechanical properties have their origin in the properties of the individual filaments and their connectedness, like cross-linking geometry and pore size distribution. While the macroscopic properties of biopolymer networks have been extensively studied, there has been a lack of experimental techniques that can simultaneously determine mechanical and architectural properties of networks in situ with single filament resolution. This work introduces Thermal Noise Imaging (TNI) as a novel quantitative method to address these issues. TNI is a three-dimensional scanning probe technique that utilizes the confined thermal motion of an optically trapped particle as a three-dimensional, noninvasive scanner for soft, biological material. Using a photonic force microscope (PFM) custom built for this research, the position of the probe can be detected with nanometer precision and megahertz bandwidth. Two sets of single molecule experiments are described that demonstrate the microscope's exceptional precision and stability. Micrometer scale thermal noise images inside a collagen network are shown and quantitative information about cross-linking geometry is extracted from the data. Further, by imaging microtubules grafted to a support it is shown that the acquired data yield information about the transversal fluctuations of the imaged fibers and about fiber elasticity. These results pave the way for an investigation of force distributions inside biopolymer networks on the single filament level.Item Supported Lipid Bilayer Electrophoresis: A New Paradigm in Membrane Biophysics and Separations(2012-11-28) Pace, Hudson 1982-The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind?s understanding of the cellular membrane.Item Ultra-precise manipulation and assembly of nanoparticles using three fundamental optical forces(2012-12) Demergis, Vassili; Florin, Ernst-Ludwig; Shubeita, George T; Fink, Manfred; Makarov, Dmitrii E; Korgel, Brian AThe invention of the laser in 1960 opened the door for a myriad of studies on the interactions between light and matter. Eventually it was shown that highly focused laser beams could be used to con fine and manipulate matter in a controlled way, and these instruments were known as optical traps. However, challenges remain as there is a delicate balance between object size, precision of control, laser power, and temperature that must be satisfied. In Part I of this dissertation, I describe the development of two optical trapping instruments which substantially extend the allowed parameter ranges. Both instruments utilize a standing wave optical field to generate strong optical gradient forces while minimizing the optical scattering forces, thus dramatically improving trapping efficiency. One instrument uses a cylinder lens to extend the trapping region into a line focus, rather than a point focus, thereby confining objects to 1D motion. By translation of the cylinder lens, lateral scattering forces can be generated to transport objects along the 1D trapping volume, and these scattering forces can be controlled independently of the optical gradient forces. The second instrument uses a collimated beam to generate wide, planar trapping regions which can con fine nanoparticles to 2D motion. In Part II, I use these instruments to provide the first quantitative measurements of the optical binding interaction between nanoparticles. I show that the optical binding force can be over 20 times stronger than the optical gradient force generated in typical optical traps, and I map out the 2D optical binding energy landscape between a pair of gold nanoparticles. I show how this ultra-strong optical binding leads to the self-assembly of multiple nanoparticles into larger contactless clusters of well de ned geometry. I nally show that these clusters have a geometry dependent coupling to the external optical field.