Lietzow Michael A, Hubbell Wayne L
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008, USA.
Biochemistry. 2004 Mar 23;43(11):3137-51. doi: 10.1021/bi0360962.
A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.
蛋白质定点自旋标记技术发展的一个目标是将氮氧自由基侧链的运动与局部结构、相互作用及动力学相关联。利用已知结构的α-螺旋蛋白在这一目标上已取得显著进展，而本研究是对β-折叠蛋白——细胞视黄醇结合蛋白（CRBP）进行类似探索的第一步。氮氧自由基侧链沿着内部链和边缘链引入。在内部链的位点，侧链运动受到与相邻链侧链相互作用的强烈影响，产生了丰富多样的动态模式（弱有序、强有序、固定）以及受链扭转调制的复杂电子顺磁共振光谱。利用诱变技术探索了产生这些动态模式的相互作用，结果表明非氢键连接的相邻残基在产生高度有序状态方面具有特别重要的作用。在β-折叠的边缘链上，侧链运动简单且弱有序，类似于α-螺旋溶剂暴露表面的运动。提出了一个简单的工作模型，该模型可以解释所遇到的各种动态模式。总体而言，结果表明氮氧自由基侧链是侧链相互作用的有效探针，定点自旋标记应该是监测涉及β-折叠拓扑结构变化的构象变化的有力手段。
