Conformational changes of troponin C within the thin filaments detected by neutron scattering.

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered "invisible" to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 A (99% confidence limits between 22 A and 23 A) and 24 A (99% confidence limits between 23 A and 25 A) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 A (99% confidence limits between 49 A and 57 A) and 49 A (99% confidence limits between 44 A and 53 A) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4A is associated with large conformational changes of the entire Tn molecule by binding of Ca2+.

The structures of the active center in dark-adapted bacteriorhodopsin by solution-state NMR spectroscopy.

The two forms of bacteriorhodopsin present in the dark-adapted state, containing either all-trans or 13-cis,15-syn retinal, were examined by using solution state NMR, and their structures were determined. Comparison of the all-trans and the 13-cis,15-syn forms shows a shift in position of about 0.25 A within the pocket of the protein. Comparing this to the 13-cis,15-anti chromophore of the catalytic cycle M-intermediate structure, the 13-cis,15-syn form demonstrates a less pronounced up-tilt of the retinal C12[bond]C14 region, while leaving W182 and T178 essentially unchanged. The N[bond]H dipole of the Schiff base orients toward the extracellular side in both forms, however, it reorients toward the intracellular side in the 13-cis,15-anti configuration to form the catalytic M-intermediate. Thus, the change of the N[bond]H dipole is considered primarily responsible for energy storage, conformation changes of the protein, and the deprotonation of the Schiff base. The structural similarity of the all-trans and 13-cis,15-syn forms is taken as strong evidence for the ion dipole dragging model by which proton (hydroxide ion) translocation follows the change of the dipole.