The overall aim of this project is to determine by X-ray crystallography the precise molecular architecture of certain alpha-proteins that have dynamic as well as structural roles. Muscle proteins are a central focus and provide the background for studying related systems. We seek to determine how the myosin motor works by obtaining atomic resolution """"""""snapshots"""""""" of the molecule in different states of contraction.
Our first aim i s to extend our crystallographic studies of both scallop and vertebrate smooth muscle myosins focusing particularly on states that bind strongly to actin. We also wish to identify specific structural differences between these isoforms that may clarify their different modes of regulation. Crystallography will also be used to test a specific model we have proposed for regulation in unconventional myosins which bind calmodulin, rather than light chains. In contrast to molluscan and vertebrate smooth muscle myosins, many conventional myosins are regulated by the Ca2+-dependent troponin/tropomyosin switch.
Our second aim i s to visualize the detail the atomic structures of the tropomyosin/troponin switch which controls contraction in many conventional myosins. The current structure determination of a fragment of tropomyosin will be completed and extended to the critical head-to-tail joint region of the filament. The atomic architecture of both the skeletal and cardiac troponin complexes, where somewhat different mechanisms may be involved in control of contraction, are also being determined.
The third aim, in a related area, is the analysis of fibrinogen-fibrin assembly. We intend to improve the resolution and completeness of the structure we have obtained of almost the entire bovine fibrinogen molecule. In this effort we are focusing on the central """"""""E fragment"""""""" which we have crystallized. Another goal is the crystallization of the so-called """"""""DDE"""""""" complex comprising the central region with the complementary sites of two D regions. This ternary complex is the nucleus for fibrin assembly. The final goal is the analysis of the alpha-helical coiled coil motif - especially in the large fibrous proteins whose structures we are determining by crystallographic methods. These include both canonical and non-canonical coiled coils We are convinced that detailed knowledge of the molecular mechanisms of muscle contraction and of the Cab controlled troponin/tropomyosin switch is essential in order to correct malfunctions in various muscle diseases. Without atomic structures of these muscle proteins in different physiological states the significance of disease producing mutations cannot be understood, nor can there be rational intervention at the molecular level directed to overcoming functional defects in these molecules. Similarly, in order to understand and control clot formation, as well as the interactions of fibrin with different cell types, it is essential to have detailed structural information about the molecules and their interactions in the clot. A beeper understanding of factors influencing folding, stability and partner selection in alpha-helical coiled coils will allow the design of therapeutic and diagnostic peptides targeted to naturally occurring coiled-coil motifs.
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