Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.
Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.
复制标题
肌球蛋白中的三硝基苯化反应性赖氨酸残基可在 ATP 水解的连续步骤中检测杠杆臂的运动。
DOI：
10.1021/bi990149r
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Muhlrad,A
中科院分区：
文献类型：
--
作者：
Ajtai,K;Peyser,YM;Park,S;Burghardt,TP;Muhlrad,A
骨骼肌球蛋白亚片段1（S1）中反应性赖氨酸（Lys 84）的三硝基苯化将手性探针（TNP）引入S1的催化和杠杆臂结构域的界面[Muhlrad（1977）Biochim. Biophys. Acta 493，154 - 166]。TNP-改性的S1（TNP-赖氨酸84-S1）中的TNP吸收和圆二色性（CD）光谱以及天然S1中的赖氨酸84三硝基苯化速率的特征表明ATP酶瞬变和捕获的磷酸盐类似物之间存在一一对应关系。使用S1的晶体学坐标[Rayment et al.（1993）Science 261，50 - 58]模拟磷酸盐类似物诱导的TNP-Lys 84-S1结构，在Gly 699和Gly 710处旋转以近似ATP酶期间的构象变化。的CD和吸收光谱特性的模型结构进行了比较，所观察到的模拟诱导的结构。模型计算，首先测试的三硝基苯基化的六肽与已知的结构，被施加到TNP-赖氨酸84-S1。他们表明，ATP结合引发Gly 699的旋转，而Gly 710和Gly 699的旋转伴随着产物释放前的ATP分裂。ATP酶过程中计算的杠杆臂轨迹表明：（i）核苷酸诱导的Lys 84三硝基苯基化抑制的合理机制，以及（ii）三硝基苯基化诱导的S1 Mg 2 +-和K+-EDTA ATP酶变化来自杠杆臂与TNP在Lys 84处的碰撞。TNP是S1的位点特异性结构扰动剂，并且是Lys 84修饰对动态S1结构的影响的手性报告基团。因此，TNP-Lys 84-S1相当于一种对修饰残基局部结构具有内在敏感性的基因工程突变体。
Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977)Biochim. Biophys. Acta 493, 154−166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993)Science 261, 50−58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.