Our objective is to determine the precise molecular architecture of certain alpha-fibrous proteins that have dynamic as well as structural roles in the cell. The chief methods are coordinated electron microscopy and X-ray crystallography, together with biochemistry. Muscle proteins are a central focus and provide the background for studying related systems. A major aim is t determine the structure, motions and interactions in three protein switches that control contraction. The topology of the myosin head and its associated light chains, the crystal structure of the tropomyosin/troponin complex, and the structure and interactions of the myosin rod and paramyosin are being analyzed to establish models for regulation. In each case, the structure of the individual protein components will be determined to the highest possible resolution, and the changes in structure and interactions that accompany switching will be sought. The molecular basis of blood clotting is being studied by a similar approach: the crystal structure of fibrinogen is being determined by X-ray crystallography and cryoelectron microscopy in order to understand its self-assembly into the fibrin clot. The principle of self-assembly applies to many of these proteins. This means that under appropriate conditions, these highly organized structures can be dissociated and reassembled in vitro to form ordered structures (including crystals) closely related to those found in vivo. In contrast to the ordered arrays formed by the purified proteins, however, the native structures are not so accessible to detailed analysis. The alpha-helical coiled-coil motif that characterizes tropomyosin, myosin rod and paramyosin has been shown to have a widespread occurrence in a diverse range of proteins. Using predictive methods based on known structures, we aim to define more fully the designs of other related alpha- proteins (such as dystrophin, spectrin,land alpha-actinin) which have not yet been solved crystallographically. Knowledge of the molecular mechanism of contraction and its regulation is needed to account for malfunctions in various heart and muscle pathologies. Similarly, a full understanding of blood clotting, and its malfunction in certain cardiovascular diseases, requires detailed information about the structure and interactions of the fibrinogen molecule. These studies aim to identify distinctive features of fibrous protein structure that are essential to function, and will lead to a deeper understanding of both normal and abnormal cell functions.
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