Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009. / Title from title screen (site viewed July 6, 2010). PDF text: x, 133 p. : ill. (some col.) ; 3 Mb. UMI publication number: AAT 3366064 . Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Gelsolin was labelled with the sulphydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labelling using non-denaturing conditions was 1.9 ± 0.5 acrylodans per gelsolin molecule. Circular dichroism and viscosity studies showed no significant effect on gelsolin structure and function on incorporation of the label. Circular dichroism studies did not detect Ca²⁺ effects on aerylodan-labelled gelsolin, but fluorescence studies detected subtle changes in the protein. The presence of Ca²⁺ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I⁻ and a decrease in fluorescence polarisation of the acrylodan-labelled gelsolin. These indicate an increased degree of exposure of the fluorescent label to the solvent environment on interaction of gelsolin with Ca²⁺. Actin binding to gelsolin was evident from a decrease in fluorescence intensity, an increase in sensitivity to quenching and an increase in fluorescence polarisation. Actin binding increases the exposure of the acrylodan label to solvent, as does Ca²⁺ binding. / Science, Faculty of / Chemistry, Department of / Graduate

Fluorescence spectroscopy can be used to probe protein conformation and is recognized as a technique that provides very specific information. It has been applied/ in recent years/ to the study of tropomyosin (TM) and its role in regulation of contractile processes. In this thesis, two different approaches were used to further the understanding of the structure/function relationship in the two chain coiled coil of tropomyosin. The first involves a comparative study on TM and non-polymerizable TM (NPTM) (Mak, A.S., and Smillie, L.B. (1981) Biochim. Biophys. Res. Commun., 101, 208-214). Fluorescence involving pyrene (Py) and acrylodan (AD) bound at the only cysteine residue in the molecule (Cys-190), and circular dichroism (CD) studies led to the main conclusion that, while the two species, are very similar in stability, the COOH-terminus is required to hold the Cys-190 region in a specific conformation. This long-range structural effect may play a role in regulation of contraction. A species having one intact COOH-terminus, made by hybridizing TM and NPTM, was found to be non-polymerizable suggesting that one intact COOH-terminus is insufficient to permit overlap with the NH₂-terminus of a neighbouring TM under polymerizing conditions. Unlike the TM/NPTM hybrid, the hybrid of TM and platelet TM (P-TM) was difficult to make due to the sequence mismatches in the terminal regions, but small quantities could be detected by loss of excimer fluorescence from Py-P-TM on rapid cooling of a heated mixture of Py-P-TM and cardiac TM (C-TM). The second approach was to investigate the effect of actin-binding proteins on the structure and function of tropomyosin. DNase I depolymerizes F-actin and is known to interfere with the end-to-end polymerizability of tropomyosin (Payne, M.R., Baydoyannis, H., and Rudnick, S.E. (1986) Biochim. Biophys. Acta 883, 454-459). Results presented here from fluorescence studies suggest that this effect is caused by a localized loss of structure in the tropomyosin at the sites of labelling upon binding of DNase I. This result is supported by CD studies on labelled and unlabelled tropomyosins. Gelsolin is another actin-binding protein found in many cell types and in extracellular fluids. It is shown here to be able to depolymerize tropomyosin, but its mechanism of action is not the same as that of DNase I. The effect of interaction of gelsolin on the structure of tropomyosin, as determined from fluorescence studies, is negligible. / Science, Faculty of / Chemistry, Department of / Graduate

The use of fluorescent molecules as probes of protein conformation is recognized as a technique which provides very specific information and has been applied, in recent years, to the study of the role of tropomyosin (TM) in the regulation of contractile processes. The isolation and sequencing of TM from horse blood platelets (P-TM) has shown it to be different from muscle TM, especially near the NH₂-and COOH-termini. These differences have been suggested to weaken end-to-end interaction of P-TM molecules. TM's are two chain coiled coils and P-TM has cysteine residues at the penultimate COOH-terminus position of adjacent chains. These can be labelled with sulfhydryl-specific fluorescent probes that reflect conformational changes in that region of the molecule via changes in their emission characteristics. The results of experiments on both pyrene (Py) (40) and acrylodan (AD) labelled P-TM show that there is a preferred interaction of the COOH-terminus of P-TM with the NH₂-terminus of cardiac TM over that with the NH₂-terminus of P-TM. This indicates that the altered NH₂-terminus of P-TM, with respect to muscle TM, is responsible for the relative loss of polymerizability of P-TM at low salt concentration. Addition of actin to the Py-P-TM (40) and AD-P-TM species showed changes in emission characteristics indicative of binding to the F-actin filaments, suggesting that the presence of the probes had not affected the function of the P-TM adversely. However, the presence of pyrenes at the COOH-terminus seemed to reduce further the ability of P-TM to self-polymerize. Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence polarization suggested that, contrary to the theory of Skolnick and Holtzer on the stability of two chain coiled coils, the region towards the COOH-terminus is among the last to lose its helical character. / Science, Faculty of / Chemistry, Department of / Graduate

Graduation date: 1994

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 22, 2008) Includes bibliographical references.

No description available.