Kraft T, Xu S, Brenner B, Yu L C
Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
Biophys J. 1999 Mar;76(3):1494-513. doi: 10.1016/S0006-3495(99)77309-1.
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 MgATPgammaS, 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 MgATPgammaS 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 I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS 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.
为了研究细肌丝激活时弱横桥与肌动蛋白附着的可能结构变化，在存在饱和浓度的MgATPγS（一种用于弱结合状态的核苷酸类似物）的情况下，在低钙和高钙浓度下记录了来自兔腰大肌的皮肤纤维的二维（2D）X射线衍射图。我们还研究了在离子强度高于和低于50 mM的松弛条件下记录的2D X射线衍射图，因为从溶液研究中提出，将离子强度降低到50 mM以下也会诱导细肌丝的激活。对于这个项目，必须建立一种新的制备方法，允许从单根肌肉纤维而不是天然纤维束记录2D X射线衍射图。这是为了最小化纤维内底物的消耗或产物的积累。当钙浓度升高时，用MgATPγS记录的衍射图显示出子午反射和层线强度的小变化，这部分可归因于钙与细肌丝结合的影响（I380增加，第一肌动蛋白层线强度降低，I59增加），部分可归因于弱附着横桥的小结构变化（例如，I143和I72增加）。在MgATPγS存在下，钙诱导的弱附着于肌动蛋白的横桥的小规模结构重排与我们之前在高钙条件下观察到的MgATPγS横桥附着和解离速率常数降低一致。然而，没有发现证据表明当通过添加钙激活肌动蛋白丝时，弱附着的横桥会朝着立体特异性构象改变其附着模式。同样，将离子强度降低到小于50 mM不会诱导从非立体特异性到立体特异性附着的转变。
