The structure of chicken skeletal muscle troponin C (TnC) has been refined to an R value of 0.168, using 14 788 reflections, in the resolution range 8.0-1.78 .~,. Our earlier 2 ,A, resolution structure [Satyshur, Rao, Pyzalska, Drendel, Greaser & Sundaralingam (1988).J. Biol. Chem. 263, 1628-1647 served as the starting model. The refined model includes atoms for all protein residues (1-162), 2 Ca 2+ ions, 169 water molecules and one sulfate ion. The high-resolution refinement shows more clearly the details of the protein and water structure. The side chains Glu63, Cysl01, Arg123, Aspl40 and Asp152 adopt two discretely ordered conformations. The long central helix is only slightly curved/bent (7.9 ° ) and all the central helix NH-.-O--C hydrogen bonds are intact. Seven of the nine carbonyl O atoms of the mid segment of this helix, including the D/E linker region, are hydrogen bonded to water molecules which weakens the helix hydrogen bonds. In contrast, in each of the protected upper and lower thirds of the long central helix, only two carbonyl O atoms are hydrogen bonded to water molecules. The hydrogen-bonding patterns displayed by some of the carbonyl O atoms of NT and A helices of the N-terminal domain and the F and H helices of the C-terminal domain, which are on the exposed surface of the protein, are similar. The B helix of the calcium-free site I is kinked, with the local helix axes at either end making an angle of 39 ° , by two inserted water molecules between N--H and O--C groups, breaking the adjacent helix hydrogen bonds. A sul-

The apparent length dependence in the calcium sensitivity of tension development in skeletal muscle has been investigated in the present study. At sarcomere lengths of 2.46-2.62 micron, the Hill plot of tension-pCa data is well fit by not one but two straight lines, suggesting the possible involvement of more than a single class of Ca2+-binding site in tension development. On the other hand, increasing the sarcomere length to 3.00-3.25 micron yielded Hill plots that were described by a single straight line, which indicates that at long lengths tension might be regulated by the binding of Ca2+ to a single class of Ca2+-binding sites, presumably the low affinity sites of TnC. This length-dependent transformation of the tension pCa relation occurred at free Mg2+ concentrations of both 0.05 and 3.2 mM. Although the mechanism of this effect is uncertain, plausible explanations for the biphasic Hill plot at the shorter lengths include the possible involvement of Ca2+ activation of the thick filaments and/or myosin LC2 phosphorylation in the process of tension development.

The frequency dependencies of the storage and loss shear moduli, G' and G", of myosin rod solutions at 1.0 and 7.0 degrees C were measured by use of the Birnboim-Schrag multiple lumped resonator apparatus in solvents with and without glycerol. The infinite dilution moduli were determined and compared with theoretical models for a rigid rod and a freely jointed trinodular rod and with an empirical model for a semiflexible rod. Only the latter could fit the data. A rotational relaxation time of 25 mus and a slowest bending time of 3.1 mus, both reduced to water at 20 degrees C, were determined from the fit. A persistence length of about 130 nm was obtained from either the bending time, the rotational relaxation time, or the intrinsic viscosity. The average thermal excursion of the end of subfragment 2 was estimated to be 26 nm, more than sufficient to span the gap between the thick and thin filaments in muscles at all sarcomere lengths. Thus, a hinge between heavy meromyosin and light meromyosin does not appear necessary for myosin-actin contact. Young's modulus of about 1 x 10(9) N/m2 also makes it unlikely that subfragment 2 can be the elastic element in the Huxley-Simmons model of muscle contraction.

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