No description available.

Infrared light (IR) was first discovered by Friedrich Wilhelm Herschel in 1800. However, until 1940’s, molecular IR studies involved only water and small organic molecules, because of the long measurement times. Development Fourier transform infrared spectroscopy (FTIR) has minimized the time required to obtain data, making it possible to investigate bigger biological systems, e.g. proteins and nucleic acids.This thesis concentrates on the applications of different IR spectroscopic techniques to a variety of biological systems and development of new approaches to study complicated biological events. The first paper in this work concerns using so-called caged compounds to study the aggregation of Alzheimer’s Aβ-peptide which is linked to the formation of neurotoxic fibrils in the brain. By adding caged-sulfate to the Aβ samples we were able to change the pH of the sample, while recording IR data and study fibril formation in a time-resolved manner. Then we used caged–ADP to study the production of ATP and creatine, mediated by creatine kinase (CK). Using CK as a helper enzyme we studied the effects of the phosphate binding on the secondary structure of SR Ca2+ATPse and determined the structural differences between two similar states Ca2E1ADP and Ca2E1ATP. In the second part of the thesis we used ATR-FTIR spectroscopy and a specially designed dialysis setup, to develop a general method to detect ligand binding events by observing the IR absorbance changes in the water hydration shell around the molecules. The same method was used to determine the binding of DNA to the transcription factors of the E2F family. E2F proteins play main part in the gene regulatory networks that control cell development. However how they recognize their DNA-binding sites and the mechanism of binding is not well understood. By using ATR-FTIR, we observed the changes in the secondary structure of the proteins, as well as the distortions to the DNA upon E2F-DNA complex formation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

No description available.

<p>While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment. </p><p>The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment. </p><p>The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.</p> / Dissertation

The phosphine crown ether, 16-(4'diphenylphosphinophenyl)-1,4,7,10,13-pentaoxa-16azacyclooctadecane (III), was synthesized using a reaction scheme beginning with n-phenyldiethanolamine and the dichloride of tetraethylene glycol, with an overall yield of 4%. Platinum and Palladium complexes of the ligand, of the form MC12L2, were synthesized as well. 13C NMR and picrate extraction data indicate III and IV (the crown-5 analog) both moderately bind sodium (14%, 15%) and potassium ions (17%, 28%). Compound V (a crown-5, triphenylphosphine-based ligand) will bind both sodium and potassium ions as well (18%, 6%). When IV is complexed to nickel carbonyl (Ni(CO)3), the addition of sodium and potassium ions cause the Al carbonyl stretching frequency to increase slightly (0.3 cm-1, 0.2 cm 1). For comparison, the addition of a proton causes the A1 carbonyl stretching frequency to increase 5.2 cm-1. However, the shift in the A1 carbonyl stretching frequency upon the addition of sodium or potassium ions indicates that ion binding by the crown ether is communicated to the phosphorus and finally to the carbonyl groups.Ball State UniversityMuncie, IN 47306 / Department of Chemistry

This work describes the results of experimental study of the flow of soft objects (microgels) through microchannels. This work was carried with the intention of building a fundamental biophysical model for the flow of neutrophil cells in microcirculatory system. In Chapter 1 we give a summary of the literature describing the flow of cells and “model cells” in microchannels. Paramount to this we developed methods to modify microchannels fabricated in poly(dimethyl siloxane) (PDMS). Originally, these microchannels could not be used to mimic biological microenvironments because they are hydrophobic and have rectangular cross-sections. We designed a method to create durable protein coatings in PDMS microchannels, as outlined in Chapter 3. Surface modification of the channels was accomplished by a two-step approach which included (i) the site-specific photografting of a layer of poly(acrylamide) (PAAm) to the PDMS surface and (ii) the bioconjugation of PAAm with the desired protein. This method is compatible with different channel geometries and it exhibits excellent longevity under shear stresses up to 1 dyn/cm. The modification was proven to be successful for various proteins of various molecular weights and does not affect protein activity. The microchannels were further modified by modifying the cross-sections in order to replicate cardiovascular flow conditions. In our work, we transformed the rectangular cross-sections into circular corss-sections. Microchannels were modified by polymerizing a liquid silicone oligomer around a gas stream coaxially introduced into the channel, as outlined in Chapter 3. We demonstrated the ability to control the diameter of circular cross-sections of microchannels. The flow behaviour of microgels in microchannels was studied in a series of experiments aimed at studying microgel flow (i) under electrostatic interactions (Chapter 4), (ii) binding of proteins attached to the microgel and the microchannel (Chapter 5) and (iii) under the conditions of varying channel geometry (Chapter 6). This work overall present’s new methods to study the flow of soft objects such as cells, in the confined geometries of microchannels. Using these methods, variables can be independently probed and analyzed.

Conformational selection is the idea that proteins traverse positions on the conformational space represented by their potential energy landscape, and in particular positions considered as local energy minima. Conformational selection a useful concept in ligand binding studies and in exploring the behavior of protein structures within that energy landscape. Often, research that explores protein function requires the generation of conformational ensembles, or collections of protein conformations from a single structure. We describe a method of conformational ensemble generation that uses joint-constrained rigid-body dynamics (an approach that allows for explicit consideration of rigidity) and the elastic network model (providing structurally derived directional guides for the rigid-body model). We test our model on a selection of unbound proteins and examine the structural validity of the resulting ensembles, as well as the ability of such an approach to generate conformations with structural overlaps close to the ligand-bound versions of the proteins.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 190-225).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [96]-100).