Motion of spin-labeled side chains in T4 lysozyme, correlation with protein structure and dynamics
Motion of spin-labeled side chains in T4 lysozyme, correlation with protein structure and dynamics
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DOI：
10.1021/bi960482k
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发表时间：
1996-06-18
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Mchaourab, HS;Lietzow, MA;Hubbell, WL
为了系统地研究硝基氧侧链迁移率与蛋白质结构之间的关系，我们制备了 30 个 T4 溶菌酶的单半胱氨酸取代突变体，并用硫氢基特异性硝基氧试剂进行自旋标记。根据生物活性、圆二色性和折叠自由能的变化来评估由氮氧氨基酸取代天然残基引起的扰动。硝基氧在稳定性和活性方面产生了上下文相关的变化，类似于在同一位点用天然氨基酸取代所观察到的变化，但对圆二色性光谱影响很小。在溶剂暴露位点，结构扰动在主链折叠水平上似乎很小。氮氧侧链迁移率忠实地反映了所有研究位点的蛋白质三级折叠。硝基氧侧链迁移率的主要决定因素是三级相互作用和主链动力学。三级相互作用将侧链的流动性限制在与相互作用程度密切相关的程度上。在螺旋间环位点，侧链具有高迁移率，与高晶体学热因素一致。在α-螺旋的暴露表面上，侧链迁移率不受与最近邻侧链相互作用的限制，而是由主链动力学决定。一个意想不到的结果是螺旋 C 端和 N 端附近残基的迁移率之间存在显着差异。这些结果为定点自旋标记实验中的另一个维度的信息提供了基础，这些信息可以根据蛋白质三级折叠、其平衡动力学和时间依赖性构象变化来解释。
Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra, At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of a-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.