Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008, USA.
Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16241-6. doi: 10.1073/pnas.1111420108. Epub 2011 Sep 12.
A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.
一个二硫键连接的氮氧自由基侧链（R1）是最广泛用于确定蛋白质拓扑结构、绘制结构变化图以及通过定点自旋标记来表征纳秒级骨架运动的自旋标记。尽管 R1 的内部运动和优选的构象数受到限制，但将自旋间距测量值和空间取向信息转化为结构约束是具有挑战性的。在这里，我们引入了一种高度受限的氮氧自由基侧链 RX，作为 R1 的替代品，用于这些应用。RX 是通过双功能甲硫磺酸酯试剂与 i 和 i + 3 或 i 和 i + 4 位的半胱氨酸残基在 α-螺旋中、i 和 i + 2 位的β-折叠中或与相邻β-折叠中半胱氨酸残基之间的交叉连接反应形成的。EPR 光谱分析、T4 溶菌酶中 RX 的晶体结构以及含有 RX 的固定化蛋白质上的脉冲电子-电子双共振（ELDOR）光谱分析均揭示了侧链的高度受限内部运动。与受限的几何形状一致，RX 侧链对之间的自旋间距分布比类似的 R1 对更窄。作为 RX 内部运动受限的重要结果，通过 ELDOR 检测到的光谱扩散揭示了蛋白质的微秒内部运动。总的来说，这些数据表明，RX 侧链将有助于通过 EPR 光谱进行距离映射、确定定向样品中螺旋段的空间取向以及测量微秒时间尺度上的结构波动。
