Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: A site-directed spin-labeling study
Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: A site-directed spin-labeling study
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DOI：
10.1021/bi960849l
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发表时间：
1996-09-24
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：
Altenbach, C;Yang, K;Hubbell, WL
用巯基特异性氮氧化物试剂对牛视紫红质Q225-I256氨基酸进行了30个连续的单半胱氨酸取代突变体的修饰。该序列包括E-F螺旋间环，一个转导蛋白相互作用位点。附加的氮氧化合物与亲水性和疏水性顺磁探针在溶液中的碰撞accessands进行了测定，和侧链流动性方面的电子顺磁共振光谱分析，无论是在黑暗中和光活化后。可及性数据显示视紫红质多肽链在V227-K231范围内并且再次在V250-V254范围内穿过水/疏水边界。在疏水段中，可及性和流动性数据都与螺旋结构一致。在位于水相中的序列的区域中，自旋标记侧链的可及性和流动性的周期性变化表明E-F螺旋间环主要是α-螺旋的，由E和F螺旋分别约1.5和3圈的规则延伸形成。从氮氧自由基迁移率判断，螺旋E在水相中的延伸比螺旋F的延伸更动态。自旋标记的视紫红质的光活化后的电子顺磁共振特性的变化表明，发色团异构化的结果，可以解释的螺旋延伸到水循环区域的运动方面的结构变化的模式。
Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra analyzed in terms of side chain mobility, both in the dark and after photoactivation. Accessibility data shows that the rhodopsin polypeptide chain crosses an aqueous/hydrophobic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessibility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regular extensions of the E and F helices by about 1.5 and 3 turns, respectively. Judging from nitroxide mobilities, the putative extension of helix E in the aqueous phase is more dynamic than that of helix F. Changes in the electron paramagnetic resonance characteristics of the spin-labeled rhodopsin upon photoactivation indicate that chromophore isomerization results in patterns of structural changes that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.