Browsing by Subject "Structural Biology"

Now showing 1 - 3 of 3
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    Elucidating the Structural Dynamics of Muscle Myosin Using Novel Methods in Electron Paramagnetic Resonance
    (2016-11) Binder, Benjamin
    Muscle contraction is fundamentally driven by an interaction between two proteins: actin and myosin. Myosin is a molecular motor, and assumes the active role in this relationship, coupling energy from hydrolysis of ATP with conformational changes to generate force on actin. In the context of a muscle fiber, this force causes filaments of myosin and actin to slide past one another in an ordered lattice, drawing the ends of individual contractile units (called sarcomeres) together. Concerted shortening of sarcomeres along the length of a fiber results in large-scale shortening of the entire fiber. Although muscle myosin has been the focus of intense study for many years, crucial details regarding its mechanism remain unknown. In particular, few structures of actin and myosin together have been reported—this is largely due to the inherent difficulties of handling large, filamentous protein complexes in traditional methods for structure determination. Myosin's interactions with actin are absolutely essential for macroscopic function, and this lack of structural information has created a knowledge gap: there is an abundance of functional and kinetic data for myosin in both normal and pathological states, but often no direct insight into the underlying structural causes for the observed behavior. In the present work, I seek to address this knowledge gap by providing high-resolution insight into the structural states of actin-bound myosin. My work is based on the hypothesis that allosteric coupling in myosin's catalytic domain (the domain responsible for actin binding, ATP hydrolysis, and initiation of force-generating conformational change) is accomplished via subtle internal rearrangements of individual structural elements. Furthermore, I hypothesize that these changes can be detected and quantified by innovative applications of site-directed spectroscopy. In Chapter 4, I establish a method using electron paramagnetic resonance (EPR) of a bifunctional spin label to probe nucleotide-dependent changes in the actomyosin complex. In Chapter 5, this method is expanded to include two complementary EPR techniques, ultimately providing sufficient constraints for direct modeling of nucleotide-dependent changes. Following these results, Chapter 6 addresses the ongoing development and further application of these methods within myosin and other protein systems.
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    Proteasome activation by the 19S regulatory particle: structural dynamics of 26S assembly and substrate recognition
    (2013-06) Ehlinger, Aaron Christopher
    Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes ranging from cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the last decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, a large number of both permanent and transient RP components with specialized functional roles are critical for proteasome function. This research investigates the dynamic nature of protein-protein interactions involved in proteasome assembly and substrate recruitment, and how they provide context to our current understanding of proteasome activation by the RP.
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    Structural Characterization of Sarcolipin by Solid State NMR and Investigation of its Role in the Regulation of Sarco(endo)plasmic Reticulum Calcium Adenosine-Triphospatase
    (2014-02) Mote, Kaustubh
    Structural characterization of membrane proteins and their complexes is an important and ever growing challenge to the classical techniques of biomolecular structural characterization. Rapid developments in the field of solid state NMR (ssNMR) have opened up an exciting new alternative to X-ray crystallography, as these studies can now be performed in fully hydrated lipid bilayers that faithfully mimic the physiologically relevant conditions. Nonetheless, routine application of ssNMR on biomolecular systems is hampered by their low sensitivity and spectral resolution. In this work, we have addressed these challenges by developing new strategies to study membrane proteins by ssNMR. With a set of improved pulse sequences for oriented and magic angle spinning techniques in ssNMR, we determined the topology (i.e. the structure and transmembrane orientation) of sarcolipin, a regulator of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA), in lipid bilayers. These techniques are further used to study the complex between these two proteins and understand the molecular basis for this regulatory interaction. The methodological developments reported here are transferable to studies on other membrane proteins and they clear several roadblocks in the successful application of ssNMR for these challenging bio-molecular systems. Finally, we present how these studies have furthered our understanding of the regulation of muscle relaxation process by SERCA. These findings represent the first steps in designing new therapeutic approaches for cardiac and skeletal muscle disorders.