Structural origin of weakly ordered nitroxide motion in spin-labeled proteins
Structural origin of weakly ordered nitroxide motion in spin-labeled proteins
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DOI：
10.1002/pro.96
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发表时间：
2009-05-01
期刊：
影响因子：
8
通讯作者：
Hubbell, Wayne L.
中科院分区：
文献类型：
--
作者：
Fleissner, Mark R.;Cascio, Duilio;Hubbell, Wayne L.
用于蛋白质定点自旋标记的二硫键连接的氮氧化物侧链（R1）通常在形貌多样的表面位点（包括螺旋、环和β-折叠的边缘链上的那些）处表现出弱有序的z轴各向异性运动的EPR谱特征。为了阐明这种运动的起源，第一晶体结构的R1，显示简单的Z轴各向异性运动在溶剂暴露的螺旋网站（131和151）和环网站（82）在T4溶菌酶已被确定。在低温或环境温度下测定的131 R1和151 R1的结构揭示了残基内C-alpha-H中心点S-delta相互作用，该相互作用固定了二硫化物基团，与R1的内部运动由围绕两个末端键的旋转支配的模型一致（哥伦布，Kalai，Jeko，Hideg和Hubbell，Biochemistry 2001; 40：3828-3846）。值得注意的是，131 R1侧链同样地填充两种旋转异构体，但EPR谱反映了单一的主导动力学群体，表明这两种旋转异构体具有由共同的二硫键-主链相互作用决定的相似的内部运动。环残基82 R1的各向异性运动也是由共同的二硫键-主链相互作用引起的，表明这种相互作用不需要特定的二级结构。如果上述观察结果被证明是普遍的，那么R1在非相互作用溶剂暴露的螺旋和环位点的顺序和速率的显著变化可以被分配给主链运动，因为内部运动基本上是恒定的。
A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of beta-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites ( 131 and 151) and a loop site ( 82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C-alpha-H center dot center dot center dot S-delta interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds ( Columbus, Kalai, Jeko, Hideg, and Hubbell, Biochemistry 2001; 40: 3828-3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.