X-RAY DIFFRACTION EVIDENCE THAT CROSSBRIDGES WEAKLY BOUND TO ACTIN IN MUSCLE ARE DISORDERED (NONSTEREOSPECIFIC BINDING). L.C.⁺ Yu, S. Xu⁺, S. Malinchik⁺, T. Kraft⁺ and B. Brenner^*. ⁺NIAMS, NIH; ^*Hannover Medical School, Hannover, Germany

Generation of isometric force, different from quick tension recovery, was proposed to result from a structural change in the actomyosin (cross bridge) complex associated with the transition from a weakly bound configuration to a strongly bound configuration (Brenner, et al., 1995). Structurally, we proposed that the weak to strong transition is from nonstereospecific attachment to stereospecific attachment. Recently we have systematically obtained two dimensional X-ray diffraction patterns from skinned rabbit psoas fiber bundles, relaxed and in rigor, such that direct quantitative comparisons can be made. (Data obtained on EMBL beamline X-13 at DESY). For 4deg.C - and 20deg.C, and ionic strength 50-150 mM, under relaxing condition, at least 6 orders of thick filament based layer lines are visible. Analysis of the axial centroid position of the inner region of the first layer line suggests that the layer line is a mixture of overlapping thick and thin filament based layer lines and their relative contribution varies with ionic strength, correlating with the fraction of weakly bound crossbridges. The behavior of the mixed layer line could be explained by modeling attachment of myosin heads to specific sites on actin while the binding angle is variable. Another evidence of nonstereospecific binding is that the diffuse scattering is little affected by considerable change in the fraction of weakly bound cross-bridges as ionic strength is changed; i.e. the weakly bound cross-bridges are disordered. As a comparison, in transition from relaxed condition to rigor where the cross-bridge attachment is known to be stereospecific, the diffuse background decreases significantly. The results support the idea that the weakly attached cross-bridges assume nonstereospecific conformations.

Some of the cross bridges that are not attached to actin form a helix around the backbone of the thick filament. The helical structure is highly sensitive to temperature. Results of the analysis and modelling will be presented. (NATO grant 930448; DFG Br849/1-4).