The Effect of Thin Filament Activation on the Attachment of Weak Binding Cross-Bridges: A Two-Dimensional X-Ray Diffraction Study on Single Muscle Fibers

Abstract

To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATP γS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATP γS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I 380, decrease in first actin layer line intensity, increase in I 59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I 143 and I 72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATP γS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATP γS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment.