Kliche W, Pfannstiel J, Tiepold M, Stoeva S, Faulstich H
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Biochemistry. 1999 Aug 10;38(32):10307-17. doi: 10.1021/bi990615c.
A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].
制备了一系列硫醇特异性交联试剂，用于研究兔骨骼肌肌球蛋白亚片段1中SH1（半胱氨酸（707））和SH2（半胱氨酸（697））之间的交联动力学。这些试剂的类型为RSS(CH(2))(n)()SSR，其中R = 3-羧基-4-硝基苯基，n = 3、6、7、8、9、10和12，跨越的距离为9至20埃。通过测量2-硝基-5-硫代苯甲酸酯的释放，用分光光度法监测反应。分别通过K(+)(EDTA)-ATP酶活性的降低和Ca(2+)-ATP酶活性的降低来测量SH1修饰的反应速率（k(1)）和交联反应速率（k(2)），并对C(n)的不同反应性进行校正。在存在和不存在MgADP的情况下，交联速率对试剂长度表现出相似的依赖性：n = 3或n = 6时的交联速率接近n = 0（埃尔曼试剂）时的速率，而n = 7和8时的交联速率则显著增加。因此，在两种状态下SH1和SH2之间的距离似乎相等，基于拉伸构象中n = 8的试剂长度，可估计为≥15埃。在严格条件下，SH1的反应性与存在MgADP时显著不同，推测是由于通过亲脂结构域的屏蔽作用。同样，在不存在MgADP时，C(3)、C(6)和C(7)的交联速率k(2)约比存在MgADP时低15倍，表明SH2区域的结构变化取决于核苷酸结合。根据最近的S1和S1-MgADP的X射线结构[雷蒙特等人（1993年）《科学》261卷，50 - 58页；古利克等人（1997年）《生物化学》36卷，11619 - 11628页]对结果进行了讨论。
