Call number: LD2668 .T4 1984 D44 / Master of Science

Methods are presented to acquire data from analytical ultracentrifugation experiments by computer using the absorption optical scanning system of the Beckman Model-E ultracentrifuge. A computer program was written which analyzes sedimentation velocity experiments by the van Holde - Weischet method and by the second moment method. The van Holde - Weischet method allows a high resolution analysis of sedimentation velocity data by eliminating the effects of diffusion on the shape of the moving boundary to provide sedimentation coefficients for a heterogeneous composition of a sample. The second moment method obtains the sedimentation coefficient by calculating the second moment point, by which the sedimentation coefficient is defined. Since it is impractical to manually analyze sedimentation velocity data by this method, these computer programs make an important analysis method available to the researcher. Using this computer program, it is now possible to analyze data to a higher resolution and accuracy than manual analysis of stripchart recordings would permit. Moreover, the time required for the analysis is greatly reduced. Data from sedimentation equilibrium experiments are analyzed by x² minimization. Further, a program was written for the acquisition of data to measure diffusion coefficients from quasi elastic light scattering experiments with a Langley Ford correlator. The analysis of autocorrelation spectra from light scattering experiments is performed by the Levenberg - Marquardt method, which allows fitting of data to nonlinear models. The model used allows the analysis of multicomponent systems by fitting to a sum of exponentials and a baseline. Traditional analysis of autocorrelation data by hand was limited to least squares fitting of the data to a linear model of one component without an optimized baseline, often an unrealistic approximation of the system. Analysis of autocorrelation data by nonlinear curve fitting increases both the accuracy and amount of data that can be analyzed. The development of the PPOL-1 208-n series of plasmids and of the miniplasmid pMX is described. These plasmids were designed to allow studies of in vitro transcription and chromatin structure after reconstitution with histones. The plasmids themselves were analyzed by sedimentation and diffusion studies using the computer programs. Sedimentation data is presented which suggests a new method for rapid estimation of S₀ (the sedimentation coefficient at zero concentration) for molecules which show a concentration dependency of the sedimentation coefficient. This is accomplished by linearly extrapolating van Holde Weischet distributions to zero concentration. Manual analysis of sedimentation velocity experiments to determine nonideality contributions required several experiments, computer analysis can provide this information in a single experiment due to the increased resolution of the method. Diffusion data for this plasmid DNA is used to demonstrate the feasibility of the multicomponent analysis presented here. Also, sedimentation measurements were carried out on reconstituted chromatin and on the effects of ethidium bromide on reconstituted chromatin. The programs were used to demonstrate significant changes in chromatin structure upon ethidium bromide binding. These changes involved the reduction of S of reconstituted plasmids upon addition of ethidium bromide as well as a reduction of heterogeneity of the sample. The data indicates the possibility of a forced exchange of nucleosomes between plasmids, as well as conformational changes in the chromatin structure. / Graduation date: 1993

This thesis covers a wide variety of projects within the domain of computational structural biology. Structural biology is concerned with the molecular structure of proteins and nucleic acids, and the relationship between structure and biological function. We used molecular modeling and simulation, a purely computational approach, to study DNA-linked molecular nanowires. We developed a computational tool that allows potential designs to be screened for viability, and then we used molecular dynamics (MD) simulations to test their stability. As an example of using molecular modeling to create experimentally testable hypotheses, we were able to suggest a new design based on pyrrylene vinylene monomers. In another project, we combined experiments and molecular modeling to gain insight into factors that influence the kinetic binding dynamics of fibrin "knob" peptides and complementary "holes." Molecular dynamics simulations provided helpful information about potential peptide structural conformations and intrachain interactions that may influence binding properties. The remaining projects discussed in this thesis all deal with RNA structure. The underlying approach for these studies is a recently developed chemical probing technology called 2'-hydroxyl acylation analyzed by primer extension (SHAPE). One study focuses on ribosomal RNA, specifically the 23S rRNA from T. thermophilus. We used SHAPE experiments to show that Domain III of the T. thermophilus 23S rRNA is an independently folding domain. This first required the development of our own data processing program for generating quantitative and interpretable data from our SHAPE experiments, due to limitations of existing programs and modifications to the experimental protocol. In another study, we used SHAPE chemistry to study the in vitro transcript of the RNA genome of satellite tobacco mosaic virus (STMV). This involved incorporating the SHAPE data into a secondary structure prediction program. The SHAPE-directed secondary structure of the STMV RNA was highly extended and considerably different from that proposed for the RNA in the intact virion. Finally, analyzing SHAPE data requires navigating a complex data processing pipeline. We review some of the various ways of running a SHAPE experiment, and how this affects the approach to data analysis.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.