Thesis (M.Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 133-138). Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2004. / Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Ligand binding sites in proteins generally include "hot spots" that contribute a large fraction of the binding free energy and, therefore, are of prime interest in drug design. To find hot spots on the protein surface, a protein can be screened against libraries of small organic molecules to identify interaction sites using nuclear magnetic resonance (NMR) spectroscopy or the X-ray crystallographic technique Multiple Solvent Crystal Structures (MSCS). Small organic molecules can bind at several locations on the surface of a protein, but many different molecules congregate only in "consensus sites" identifying the hot spots. The mapping algorithm FTMAP is a computational analogue of experimental fragment screening methods. The principles of computational mapping were used for the development and testing of the binding site identification algorithm FTSITE, implemented as a web-based server. Finding ligand binding sites in silico is a classical challenge, and the success rate of identifying the ligand binding site as the first predicted site has increased to 83% during the last decade. FfSITE, based on biophysical modeling of protein-ligand interactions, increased the success rate to 94% on the same established test sets. Critical to the success of FfSITE is the use of multiple small molecules as probes; screening by X-ray crystallography and NMR spectroscopy had demonstrated a tendency of ligand binding sites to bind small organic compounds ranging 1n shapes, sizes, and polarities. Further, FfSITE does not use surrogate measures of ligand binding propensity such as site geometries and dimensions. It was shown that FTSITE can also successfully identify allosteric ligand binding sites that can serve as candidates for drug design. Furthermore, the hot spot information provided by FfMAP was shown to guide the development of core fragments, found by experimental fragment screening , into optimal ligands for a number of drug target proteins. Computational mapping can also be used for fragment-based drug design by finding fragments with preference for some regions of the binding site. To facilitate this analysis , a server enabling the fast generation of force field parameters for user-specified small molecules or fragments was developed. / 2031-01-02

A novel chiral helical imprinted polymer (MIP) has been prepared by bulk copolymerization of 3-methyl-4-vinylpyridine and a crosslinker using chiral S-mandelic acid as template. The resulting MIPs after removing the template show optical activity that is derived from the presence of chiral helical structures of poly(3-methyl-4-vinylpyridine) (P3M4VP) embedded within the MIP matrix. The basic functional monomer 3-methyl-4-vinylpyridine (3M4VP) was utilized with the acidic template to ensure good interaction between the template and the polymer. Divinylbenzene (DVB) and ethyleneglycoldimethacrylate (EGDMA) with widely varying degree of rigidity, flexibility, and polarity were selected as crosslinking agents. Using two different crosslinkers, it was possible to optimize the MIP for maximum specificity and selectivity. Spectroscopic methods such as FT-IR and 1H NMR were used to investigate the interaction between template and functional monomer. Scanning electron microscopy and nitrogen sorption analysis showed significant difference of the surface morphological characteristics between imprinted and non-imprinted polymers. Thermogravimetric analysis (TGA) was carried out to investigate thermal stability of MIPs and NIPs. The binding studies and the selectivity of the polymers were analyzed using UV-visible spectroscopy. The chiral secondary or higher structural ordering within the MIPs was examined using circular dichroism (CD) spectrometry. The polymer preparations were evaluated and compared with non-imprinted polymers for their ability to bind the template which is a measure of the specificity of the imprinted system. The nature and degree of crosslinking and other parameters like concentration of the template solution, solvent, and time which influences the binding of these imprinted polymers towards the print molecule were also investigated to optimize the imprinted system. The ability of the S-enantiomer imprinted polymer to bind the R-enantiomer was investigated. The separation and selectivity factors have been quantified. The present study develops a successful strategy for preparing chiral MIPs, which are expected to find vital applications in chiral separation, and enantioselective release of chiral drugs.

Previous studies with conjugates containing the NH2-terminal and COOH-terminal antigenic determinants of oxidized ferredoxin from C. pasteurianum indicated a need for at least two determinants to stimulate DNA synthesis in sensitized lymphocytes. This suggested a mechanism involving cell cooperation, a possibility which has been investigated here by selectively inactivating cells binding one or the other of the determinants. Cells from immunized guinea pigs were tested in vitro for their capacity to bind antigen or to be stimulated by it before and after "antigen suicide" with radioiodinated conjugates containing the NH2-terminal or COOH-terminal determinants of oxidized ferredoxin. A microculture system for assessing antigen induced stimulation of 3H-thymidine uptake by lymphocytes was developed for this work. The data show that: 1) Lymphocytes from unimmunized guinea pigs bind both NH2-terminal and COOH-terminal determinants at a frequency of about 10-4. In immune animals the proportion of antigen binding cells increased about 4-6 fold. The frequency of cells binding the determinants depends markedly on the specific activity of antigens employed. 2) Both T and B lymphocytes bind the antigenic determinants from oxidized ferredoxin. 3) Specific inactivation of cells binding either determinant was achieved by antigen suicide with ¹²⁵I-NH₂-terminal or ¹²⁵I COOH-terminal s-BSA conjugates. Synergy occurs between the NH2-terminal binding cells and COOH-terminal binding cells in the proliferative response of sensitized lymph node cells challenged with oxidized ferredoxin in vitro. Evidence from B cell depletion studies indicates that this is a T cell-T cell interaction. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Properties of the regulatory nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. Previously, several lines of evidence have indicated the existence of both catalytic and regulatory nucleotide binding sites on the same polypeptide species. The present study concentrates on the interaction of the ATP analogue, 2'-3'-0-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate, (TNP-ATP), with sites on the non-phosphorylated and phosphorylated enzyme. In particular those conformational transitions linking TNP-ATP fluorescence to the phosphoenzyme subspecies have been sought. Previous studies have demonstrated a close relationship between TNP-ATP fluorescence and phosphoenzyme formed from ATP plus Ca²⁺, or from inorganic phosphate (Pi) in the absence of Ca²⁺, in the reverse direction of the cycle. However, the precise relationship of TNP-ATP fluorescence to the energy transducing conformations of the ATPase is controversial. TNP-ATP binding was investigated by spectrophotometric methods and by the synthesis of [ ¹⁴C] TNP-ATP. [ ¹⁴C] TNP-ATP bound to the ATPase site with high affinity ([TNP-ATP] 0. 5 = 0.12 uM), and · a stoichiometry of 5.4 nmol/mg. [ ¹⁴C] ATP binding stoichiometry was 6.1 nmol/mg, demonstrating that TNP-ATP binds to a single family of sites. The nature of the phosphoenzyme intermediate species that results in enhanced TNP-ATP fluorescence was investigated. NEM derivitization, Sr²⁺-transport and Ca²⁺-oxalate uptake have previously been found to alter the distribution or relative levels of phosphoenzyme intermediates. Modification of thiol groups responsible for phosphoenzyme decomposition (SHd), using N-ethylmaleimide (NEM) (0.4 mM) with 50 uM Ca²⁺, 1 mM AMP-PNP at pH 7.0, resulted in a 50% decrease in Ca²⁺-uptake, Ca²⁺-ATPase activity and ADP-insensitive E-P (E₂-P), while total EP (E₁-P + E₂-P = 3.2 nmol/mg), remained unaltered. ATP-dependent TNP-ATP enhanced fluorescence decreased by 50% under these conditions. Ca²⁺-oxalate induced turnover has previously been shown to decrease steady-state E₂-P levels by prevention of Ca²⁺ gradient formation. Oxalate (5 mM) caused a 40% decrease in ATP-induced TNP-ATP fluorescence levels while total EP levels remained relatively unaltered. Previous studies have shown that Sr²⁺-induced turnover favours higher levels of E₂-P by inhibiting the reverse reaction from E₂-P to E₁-P. Strontium-induced turnover increased TNP-ATP fluorescence by 10% as compared to that of Ca²⁺, without affecting steady-state E-P levels, consistent with an E₂-P conformation relationship to enhanced TNP-ATP fluorescence. The binding site for TNP-ATP on the enzyme was investigated by chase studies using millimolar concentrations of nucleotides. ATP and ADP diminished TNP-ATP fluorescence competitively, with apparent Km values of 1.25 and 0.54 mM respectively, consistent with their affinities of binding to the regulatory site. The rates of decrease of fluorescence (25 and 34 sec⁻¹ at 5 ᵒC, respectively), were of the same order of magnitude as the derived "off" rate of TNP-ATP from the site of enhanced fluorescence (33 sec⁻¹), consistent with TNP-ATP being bound to the regulatory site of the enzyme. Enhanced TNP-ATP fluorescence has previously been related to decreased water activity of the probe site. Alteration of water activity by structure- forming (Deuterium oxide) and structure-breaking solutes (KSCN) in relation to fluorescence were explored. Replacement of H₂O by D₂O altered the fluorescence of unbound TNP-ATP. The apparent for TNP-ATP binding to the E₂-P conformation of the regulatory site. The regulatory site appears to be a modified form of the phosphorylated catalytic site. It is proposed that TNP-ATP fluorescence monitors an enzyme conformation related to Ca²⁺ binding to an inward oriented site of low affinity. The mechanism of K⁺ fluorescence quenching appears to be via an acceleration of dephosphorylation, as opposed to a change in affinity of the enzyme for TNP-ATP, as previously suggested. The K⁺ sensitivity of TNP-ATP fluorescence has proved useful in demonstrating a direct interaction of valinomycin with the enzyme through the monovalent cation binding site. Valinomycin appears to bind directly to the enzyme and to selectively accelerate the "off" rate of K⁺ from this site.

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El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP. / National Institutes of Health / Revisión por pares

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).