Robert Mendelson and Edward P. Morris
Cardiovascular Research Institute and Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0130 and Biochemistry Dept., Imperial College, London SW7 2BZ
A quantitative fitting method has been used to fit the crystallographic structures of actin and myosin subfragment-one (S1) into acto-S1 cryo-electron microscopy data. Using undistorted crystallographic results, the best-fit acto-S1 structure shows some differences from the acto-S1 structure obtained from visual fitting. The best fit is produced using the F-actin model of Holmes et al. S1 residues at the acto-S1 interface are now found at a higher radius and have translated axially and rotated azimuthally. Fits using S1 with the addition of loops missing from the crystal structure were achieved using a homology search method to predict loop structures. These fits favor an arrangement in which the loop at the 50kDa-20kDa domain junction is to be found within the acto-S1 interface near the N-terminus of actin. Small rigid-body rotations and translations of the lower 50kDa domain, which improve the fit to the EM data, produce closure of the large 50kDa domain cleft and bring highly conserved residues in the lower 50kDa junction closer to actin. This lends support to the notion that the binding of ATP to acto-S1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50kDa cleft of S1.
R. A. Mendelson1, D. B. Stone1, I. Krylova1, F. Reinach2, D. Schneider3 and P. A. Timmins4
1C.V.R.I. and Dept. Biochem. & Biophys., Univ. Calif. San Francisco, CA 94143; 2Univ. Sco Paulo, Sco Paulo, Brazil; 3Brookhaven National Laboratory, Upton, NY; 4Institut Laue-Langevin, 38042 Grenoble Cedex, France.
Small-angle neutron scattering was used to investigate the radius-of-gyration (Rg) of TnI and TnC within the whole troponin complex with and without regulating concentrations of Ca2+ present. Deuterated (chicken) fast skeletal muscle TnC and (SH-less) TnI, expressed in E. coli grown on deuterated algal hydrolysate in D2O, were purified and combined with recombinant (protonated) chicken fast skeletal muscle subunits into a reconstituted whole Tn complex which displayed normal regulation of the actin-activated MgATPase of myosin subfragment 1. Scattering experiments were performed at BNL on beamline H9b (TnC) and at ILL on D11 (TnI). Samples for neutron scattering were in a 41% D2O solvent in which protonated troponin subunits (C and T or I and T) were rendered "invisible". Results show that TnI in whole Tn is a highly elongate structure. When Ca2+ binds to the TnC regulatory sites TnI undergoes a large-scale structural change [delta (Rg)/Rg= -12%] resulting in a less elongate structure. On the other hand, to date TnC in whole Tn exhibits no detectable large-scale change in structure when Ca2+ binds to its regulatory sites. Modeling of low- and wide-angle data is currently underway to elucidate the in situ shapes of TnI and TnC. Initial results show that TnI is roughly parasol shaped at low resolution.