Structure of tropomyosin at 7A resolution
Whitby, Frank G.
Phillips, George N., Jr.
Doctor of Philosophy
The crystal structure of tropomyosin has been determined by X-ray diffraction analysis at 7A resolution. Tropomyosin is a 400A-long muscle regulatory protein that consists of two parallel 33,000 Dalton alpha helices wound around one another to form a coiled coil and whose amino-acid sequence is characterized by a characteristic heptad repeat pattern. The structure was solved initially at 9A resolution by molecular replacement and refinement of a uniform wire model with a specially designed refinement procedure. Phase information was later derived from a single mercury derivative by single isomorphous replacement (SIR) refinement and used in the construction of an atomic model which was refined at 7A resolution. The model agrees well with the previous low-resolution X-ray structure and with models of tropomyosin in paracrystalline and micro-crystalline forms based on electron microscopy. The overall shape of the molecules, characteristics of the coiled coil and the geometry of interactions of molecules in the crystal are apparent from the structure. The molecules are precipitated by spermine and polymerize head-to-tail to form sheets of nearly parallel filaments, overlapping by about 2/3 of the molecular length in an antiparallel configuration. The relationship of two cysteine residues on each of the molecules was determined unambiguously by solving the structure of a mercury-labeled form of the protein. The shape of the molecule is influenced by local amino-acid sequence variations and crystal packing interactions. The inherent mobility of the molecule in the crystal indicates the importance of considering the flexibility and motions of tropomyosin in models of muscle thin filament regulation and cooperativity. The detailed structure of the head-to-tail overlap region cannot be ascertained from the present model, but will be an important focus of attention for future study. Through amino-acid sequence analysis, an element of quaternary structure, the coiled coil, can be directly predicted. However, tropomyosin is the largest of this class of proteins whose structure has actually been determined by X-ray crystallography.
Biochemistry; Biophysics; Physiology