Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (Leaves: 84-89)

Thesis (Ph.D. in Pharmacology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 145-170). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thèse (Ph. D.)--Université Laval, 2004. / Dans le titre, dans l'expression "glutaminyl-ARNtGln" les lettres Gln sont suscrites, de même que les lettres Asn dans l'expression "asparaginyl-ARNtAsn". Les légendes des ill. se trouvent sur f. en regard, avec pagination continue. Bibliogr.: f. 149-170. Publié aussi en version électronique.

Thesis (M.S.)--Florida State University, 2006. / Advisor:Thomas A. Houpt, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed June 7, 2006). Document formatted into pages; contains vi, 37 pages. Includes bibliographical references.

Thesis (M. Sc. (Pharmacy))--Rhodes University, 2006.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 161 pages. Includes Vita. Includes bibliographical references.

The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

Este trabalho teve por finalidade a implementação e desenvolvimento de novos métodos para a análise de curvas de espalhamento de raios X a baixo ângulo por sistemas monodispersos. O resultado básico final deste trabalho foi a confecção de três programas de computador e suas aplicações em proteínas de interesse biológico. ELLFIT é um programa de computador que encontra o elipsóide cuja curva de SAXS melhor se ajusta a uma dada curva experimental. Para casos favoráveis este programa é capaz de determinar a dimensão máxima e algumas características básicas do formato da partícula. CRYSOL é um programa para a avaliação de curvas de espalhamento em solução para proteínas com estrutura atômica conhecida. O programa usa a expansão de multipolos para o cálculo rápido da promediação espacial e simula uma camada de hidratação ao redor da proteína. CRYSOL pode predizer a curva de SAXS de uma determinada proteína e compará-la com dados experimentais. HOMDIM é um programa para a determinação da posição das sub-unidades de um homodímero no caso de ser conhecida somente a estrutura da sub-unidade sozinha. Dada a curva experimental e a amplitude da sub-unidade, HOMDIM procura os parâmetros posicionais que descrevem o homodímero. Estes e outros programas foram aplicados a várias proteínas. O método da expansão de multipolos foi usado na determinação do envelope molecular de uma inibidora de ALPHA-amilase. O programa CRYSOL foi utilizado para resolver uma ambiguidade na estrutura quatemária cristalina da hexocinase e o programa HOMDIM para a proposição de um novo modelo para a estrutura quatemária da aspartato transcarbamilase no estado R em solução / This work was aimed at the implementation and development of new methods for solution scattering analysis of monodisperse systems. The basic final result of this work was the development of three programs and their applications to proteins of biological interest. ELLFIT is a computer program, which finds the elipsoid whose SAXS curve has the best fit to a given experimental curve. In favorable cases, this program is able to determine the maximum dimension and some basic characteristic of the particle shape. CRYSOL is a program for evaluating the solution scattering from, proteins of known structure. The program uses multipole expansion for fast calculation of the spherically averaged scattering pattern and takes into account the hydration shell. Given the atomic coordinates it can predict the solution scattering curve and compare it with the experimental scattering curve. HOMDIM is a program to determine the position of both subunits of a homodimer when only one sub-unit structure is known. Given the experimental curve of the homodimer and the subunit scattering amplitudes. HOMDIM searches for the positional parameters, which describe the homodimer. These and other programs were used to study several proteins. The multipole expansion method was used in the shape determination of an ALPHA-amylase inhibitor. The program CRYSOL was used to solve the ambiguity in the hexokinase quaternary crystal structures and the program HOMDIM was utilized for the quaternary structure modeling of the R-state of the aspartate transcarbamilase in solution

No description available.

The concerns of the present study were to determine 1) the conditions necessary to measure displaceable [³H] L-aspartate binding to membrane fractions of the rat brain, 2) whether the binding demonstrated the charcteristics of the site which is active in vivo, and 3) whether the acidic amino acid neurotransmitters aspartate and glutamate bind to identical or different sites by comparing the pharmacological specificities of the [³H] L-aspartate binding with that of [³H] L-glutamate. The conditions of the [³H] L-aspartate binding assay were determined in synaptosomal and total particulate fractions of whole rat brain. The reaction mixture which included the membrane fraction suspended in Tris-HCl buffer (pH 7.4) in the presence or absence of the compound under test, was incubated at 37°C for 30 minutes. The reaction was stopped by centrifugation and the radioactivity in the pellet counted by liquid scintillation spectrometry. The [³H] L-aspartate binding was characterized in total particulate fractions of rat cerebellum. The apparent dissociation constant (K[sub=D]) and maximum binding (Bmax), as determined by Scatchard analysis, are 1.64 ± 0.34 μM and 7711 ± fmol/mg protein respectively. The displaceable binding is reversible, saturable, independent of the presence of NA⁺, has an affinity in the range where the neurotransmitter is active in vivo, and demonstrates a pharmacological specificity which includes stereospecificity. The compounds tested to demonstrate the pharmacological specificity were L-aspartate (IC[sub=50] = 1.81 μM), D-aspartate (IC[sub=50] = 46.6 μM), L-glutamate (IC[sub=50] = 1.24 μM), N-methyl-DL-aspartate (inactive), kainate (inactive), D-alpha-aminoadipate (inactive), and L-alpha-aminoadipate (IC[sub=50] =7.12 μM). The pharmacological specificity of [³H] L-aspartate binding was different from that of [³H] L-glutamate. When the binding data only are considered, therefore, separate receptors for aspartate and glutamate are indicated. The pharmacological specificity of the [³H] L-aspartate binding, that is the affinity of the binding site for N-methyl-DL-aspartate, D- and L-alpha-aminoadipate, however, does not correlate with the potency of these compounds derived from iontophoretic studies. L-alpha-aminoadipate is very effective while N-methyl-DL-aspartate and D-alpha-aminoadipate do not displace the [³H] L-aspartate binding. In iontophoretic studies, N-methyl-D-aspartate and D-alpha-aminoadipate are very potent as compared to aspartate while L-alpha-aminoadipate Is inactive. The [³H] L-aspartate binding then may not represent the site which is active in vivo. The characteristics of the aspartate site in vivo, however, may not be truely represented in iontophoretic studies because of, for example, uptake of the compounds. The aspartate binding site, therefore, must be identified as that which is activated in vivo. The question of separate receptors for aspartate and glutamate then must still be resolved. / Science, Faculty of / Zoology, Department of / Graduate