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螺旋束交联的设计:环氧化视黄醛的核磁共振和紫外可见光谱分析及分子模拟

Design of a helix-bundle cross-link: NMR and UV-visible spectroscopic analyses and molecular modeling of ring-oxidized retinals.

作者信息

Williams T C, Mani V

机构信息

Department of Pharmacology, Medical University of South Carolina, Charleston 29425-2251.

出版信息

Biochemistry. 1991 Mar 19;30(11):2976-88. doi: 10.1021/bi00225a035.

Abstract

In order to generate potential chemical cross-links for studying the chromophore binding site of bacteriorhodopsin and related helix-bundle proteins, MnO2 was used to oxidize all-trans-retinal's ring moiety. The structures and solution conformations of three ring-oxidized retinal analogues have been determined by using UV-visible absorption and 1H and 13C NMR spectroscopies, primarily with regard to (i) the introduction of a functional group at the ring end of the chromophore, (ii) the retention of the all-trans geometry of the polyenal side chain, and (iii) the torsional angle of the ring-polyenal bond. Analyses of their UV-visible absorption spectral parameters (lambda max, epsilon max, and vibrational fine structure) and NMR spectral parameters (1H-1H coupling constants, 1H and 13C NMR chemical shifts, and 1H homonuclear Overhauser effects) indicated the 4-oxo and the 2,3-dehydro-4-oxo derivatives both possess the twisted 6-s-cis conformation adopted by most six-membered ring analogues of retinal in solution or crystal. However, the alpha-dioxocyclopentenyl analogue exists in solution predominantly (70-80%) as the planar 6-s-trans conformer, similar to violerythrine chromophore analogues. In order to identify the minor solution forms, molecular modeling and geometry optimizations using the semiempirical molecular orbital method AM1 defined two additional symmetry-related minima at +/- 30-40 degrees in its C6-C7 torsional energy profile. Because the chromophores of bacterio- and halorhodopsins and sensory rhodopsins are bound as the 6-s-trans conformer [Harbison, G.S., Smith, S.O., Pardoen, J.A., Courtin, J.M.L., Lugtenburg, J., Herzfeld, J., Mathies, R.A., & Griffin, R.G. (1985) Biochemistry 24, 6955-6962; Baselt, D.R., Fodor, S.P.A., van der Steen, R., Lugtenburg, J., Bogomolni, R.A., & Mathies, R.A. (1989) Biophys. J. 55, 193-196], we suggest that the cyclopentenyl analogue's alpha-diketo function may be favorably positioned within the binding pocket and sufficiently reactive toward nucleophilic attack to cross-link an arginine located in or near the ring end of the chromophore cavity: Arg134 according to the current model of bacteriorhodopsin's tertiary structure [Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., & Downing, K.H. (1990) J. Mol. Biol. 213, 899-929] or Arg82 as postulated from an alternate model constructed primarily to accommodate the external point charge contribution to bacteriorhodopsin's opsin shift.

摘要

为了生成潜在的化学交联以研究细菌视紫红质和相关螺旋束蛋白的发色团结合位点,使用二氧化锰氧化全反式视黄醛的环部分。通过紫外可见吸收光谱、1H和13C NMR光谱确定了三种环氧化视黄醛类似物的结构和溶液构象,主要涉及以下方面:(i)在发色团环端引入官能团;(ii)多烯醛侧链全反式几何结构的保留;(iii)环-多烯醛键的扭转角。对其紫外可见吸收光谱参数(最大吸收波长、最大摩尔吸光系数和振动精细结构)和NMR光谱参数(1H-1H耦合常数、1H和13C NMR化学位移以及1H同核Overhauser效应)的分析表明,4-氧代和2,3-脱氢-4-氧代衍生物在溶液或晶体中均具有大多数视黄醛六元环类似物所采用的扭曲6-s-顺式构象。然而,α-二氧代环戊烯基类似物在溶液中主要(70-80%)以平面6-s-反式构象存在,类似于紫红素发色团类似物。为了识别次要的溶液形式,使用半经验分子轨道方法AM1进行分子建模和几何优化,在其C6-C7扭转能分布图中在±30-40度处定义了另外两个与对称相关的极小值。由于细菌视紫红质、嗜盐视紫红质和感官视紫红质的发色团以6-s-反式构象结合[哈比森,G.S.,史密斯,S.O.,帕尔多恩,J.A.,库尔廷,J.M.L.,卢滕堡,J.,赫茨菲尔德,J.,马西斯,R.A.,&格里芬,R.G.(1985)《生物化学》24,6955-6962;巴塞尔特,D.R.,福多尔,S.P.A.,范德斯特恩,R.,卢滕堡,J.,博戈莫尔尼,R.A.,&马西斯,R.A.(1989)《生物物理学杂志》55,193-196],我们认为环戊烯基类似物的α-二酮官能团可能在结合口袋中处于有利位置,并且对亲核攻击具有足够的反应性,能够与位于发色团腔环端或其附近的精氨酸交联:根据细菌视紫红质三级结构的当前模型[亨德森,R.,鲍德温,J.M.,切斯卡,T.A.,泽姆林,F.,贝克曼,E.,&唐宁,K.H.(1990)《分子生物学杂志》213,899-929]为Arg134,或者根据主要为适应外部点电荷对细菌视紫红质视蛋白位移的贡献而构建的替代模型假设为Arg82。

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