Myosins are molecular motor proteins involved in cell movement, vesicle and organelle transport by moving along the cytoskeletal actin filaments. They include a myosin heavy chain and at least one myosin light chain (LC). The latter are typically bilobal proteins like calmodulin, where each lobe comprises a pair of EF-hand Ca2+-binding motifs. The LCs bind to ~25-residue IQ motifs that loosely conform to an IQXXXRGXXXR consensus sequence, and impart rigidity that is crucial for myosin function. The highly motile amoeba Dictyostelium discoideum expresses seven class I myosins, two of which (MyoD and MyoB) recruit the specific LCs MlcD and MlcB, with MlcB being the first observed single-lobe LC. However, the LCs for the remaining D. discoideum class I myosins are unknown. Identifying and characterizing these LCs is one focus of this thesis, with an overall goal of understanding their role in myosin function and regulation. Nuclear magnetic resonance spectroscopy, site-directed mutagenesis, and computational modeling were used to determine the solution structure of apo-MlcB and identify the MyoB IQ motif-binding site. Apo-MlcB differs from the typical closed conformation of an EF-hand Ca2+-binding protein in the apo-state as helix 1 in its structure is splayed from the remaining helices. The MyoB IQ motif-binding surface is not altered by Ca2+, involves residues from helices 1 and 4, and from residues in the N-terminal canonical EF-hand Ca2+-binding loop, and represents a unique mode of IQ recognition by a myosin LC. Calmodulin was identified as the LC for MyoA and MyoE while another single-lobe LC, MlcC, bound to two of three IQ motifs in MyoC. The solution structure of MlcC was more similar to the C-terminal lobe of apo-calmodulin than to apo-MlcB. Chemical shift perturbation studies suggest that like apo-CaM, MlcC undergoes a global MyoC IQ motif-induced conformational change. Computational modeling of the MlcC-MyoC IQ complex indicates that this is a feasible mode of IQ recognition. The structures of MlcB and MlcC, with their different modes of IQ motif binding, provide novel insights into IQ motif binding specificity and begin to illustrate their role in myosin function and regulation. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-29 11:42:03.428

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 105-112). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [67]-75).

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Ca²⁺ sensitization of smooth muscle. Includes bibliographical references (108-130). Also available online through Digital Dissertations.

Die meisten Patienten mit hypertropher Kardiomyopathie und kongenitalen Herzerkrankungen exprimieren die atriale essentielle leichte Myosinkette (ALC-1) im Ventrikel, wo sie teilweise die ventrikuläre essentielle leichte Myosinkette (VLC-1) ersetzt. Diese VLC-1/ALC-1 Isoformveränderung korrelierte mit einem Anstieg der Zykluskinetik der Myosin-Querbrücken in chemisch gehäuteten Herzfasern aus hypertrophierten Humanventrikeln. Um die funktionelle Bedeutung der ALC-1 im gesamten intakten Herzen zu untersuchen, habe ich in der vorliegenden Arbeit ein transgenes Rattenmodell charakterisiert, das die humane ALC-1 (hALC-1) im Herzen exprimiert (TGR/hALC-1). WKY-Ratten dienten als genetisch korrekter Kontrollstamm. Mittels rekombinanter hALC-1 als Standard wurde die exprimierte hALC-1-Menge in SDS-Extrakten linker Ventrikel der TGR/hALC-1 im Western-Blot untersucht. 12 Wochen alte TGR/hALC-1 exprimierten etwa 17 mug hALC-1/mg SDS-Extrakt. Das exprimierte Transgen konnte in der Immunfluoreszenz zwischen den Z-Linien der Sarkomere lokalisiert werden. Die gerichtete Integrations des Transgens in das kardiale Myosinmolekül wurde zusätzlich noch in hochgereinigten Myosinpräparationen nachgewiesen. Analyse des linksventrikulären Proteoms durch 2D-PAGE, das zur Identifikation von etwa 3000 Proteinen führte, zeigte vergleichbare Proteinmuster in WKY und TGR/hALC-1. Die Untersuchungen der Kontraktilität des intakten isoliert perfundierten Herzen wurden mit Langendorff-Präparationen durchgeführt. Die Expression des hALC-1-Transgen führte zu statistisch signifikanten (p / Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using chemically skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the whole intact perfused-heart, a transgenic rat model overexpressing hALC-1 (TGR/hALC-1) in the heart was generated. WKY rats were used as the respective genetically correct control strain. Using hALC-1HIST protein as a standard, the amount of transgenic protein expression was quantified by Immunoblot analysis of the left ventricular tissue extracts of the transgenic rats. Twelve-week-old TGR/hALC-1 expressed around 17mug hALC-1 per mg of whole SDS-soluble protein. The transgene was localized in-between the Z-lines of the sarcomere by immunofluoresecnce microscopy. Furthermore, the proper integration of the transgene into the rat ventricular myosin was confirmed by purifying myosin from rat ventricular tissues. Whole ventricular proteome analysis by 2D-PAGE, resolved approximately 3000 proteins spots in each TGR/hALC-1 and WKY animal. The whole protein expression patterns in both animal groups showed no differences with the exception of the transgenic hALC-1 protein spot. The perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (p