Bobkov A A, Reisler E
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Biophys J. 2000 Jul;79(1):460-7. doi: 10.1016/S0006-3495(00)76307-7.
Myosin subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.
肌球蛋白亚片段1（S1），其SH1（半胱氨酸（707））和SH2（半胱氨酸（697））基团被对苯二甲酰亚胺（pPDM-S1）交联，被认为是肌球蛋白与肌动蛋白结合的弱结合状态的类似物。在本研究中，将pPDM-S1的结构特性与S1.ADP.BeF(x)和S1.ADP.AlF(4)(-)的结构特性进行了比较，即肌球蛋白弱结合状态已确定的结构类似物。为了区分SH1-SH2交联的构象效应和单功能修饰引起的构象效应，我们在实验中使用了SH1和SH2基团用N-苯基马来酰亚胺标记的S1（NPM-S1）作为对照。使用疏水性荧光染料3-[4-(3-苯基-2-吡唑啉-1-基)苯-1-磺酰胺基]苯基硼酸（PPBA）探测核苷酸口袋的状态。差示扫描量热法（DSC）用于研究S1的热稳定性。通过这两种方法，pPDM-S1的构象状态与S1.ADP.BeF(x)和S1.ADP.AlF(4)(-)复合物中未修饰的S1不同，更接近无核苷酸的S1。此外，BeF(x)和AlF(4)(-)的结合未能在pPDM-S1中诱导出与未修饰的S1中观察到的类似的构象变化。令人惊讶的是，当在S1.ADP.BeF(x)复合物上进行pPDM交联时，ADP.BeF(x)在一定程度上保护了S1的核苷酸口袋免受pPDM修饰的影响。在我们的实验中，NPM-S1的行为与pPDM-S1相似。总体而言，这项工作提供了新的证据，表明pPDM-S1的构象状态与弱结合状态类似物S1.ADP.BeF(x)和S1.ADP.AlF(4)(-)不同。SH1和SH2基团的pPDM交联及其用NPM进行的单功能标记的类似结构效应归因于这些修饰对SH1-SH2螺旋的柔韧性/流动性的抑制作用。
