PROTEIN BACKBONE STRUCTURE CHANGES THRU ACTIVE VALINE RESIDUES IN PHOTOREACTION

光反应中活性缬氨酸残基改变蛋白质主链结构

• 批准号: 6279706
• 负责人: Jin Hu
• 金额: $ 0.36万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6279706
• 关键词: biological products; biomedical resource; ear; human tissue; proteins

Evidence from x-ray, neutron diffraction and electron microscopy suggests that the Fhelix is tilted during the bR photocycle, opening the entrance of the SB region to protons and the G-helix of bR undergoes significant structural changes during the proton transport. Coincidently, 10 of the 21 valine residues in bR populate these two helices, 7 in F (Val 167, 173, 177, 179, 180, 187 and 188) and 3 in G (Va1210, 213, and 217). From Henderson's structural model for bR,681 there are two directly bonded valine-pairs (Vall79 and VaI180, VaI187 and VaI188) in the valine rich F-helix. In each of them, the carbonyls are close in space (-3 AO). The distances between the valine pairs VaI177 and VaI179 in F, A Va1210 and V11213, andVa1213 and Va1217 in G are about4.5 A to 5 Furthermore, the 13CCP/MAS NMR difference spectrum between the light-adapted (LA) state and M. photointermediate of [1-13CVal], [15N-Prol-bR shows that the chemical shifts of at least two of the 21 valine residues move upfield by at least 3.5 ppm, an indication of significant structural changes near these valine residues. This implies that these valine residues are active during the bR photocycle. Selective observation of valines 49, 69 and 199 by heteronucleaf-dipolar recoupling (REDOR) has eliminated the possibility that the three valine residues, which are not in either the F or G helices, are responsible for the changes observed in the NMR difference spectrum. Therefore, it is worthwhile to investigate if these changes occur on the valine residues in the F and G helices in the LA-+M. transition. Homonuclear dipolar recoupling (MELODRAMA) experiments were performed at a short mixing time in order to recouple only the two valine pairs located about 3 A apart and at longer mixing time to correlate the three valine pairs in the F and G helices, which are further separated. The results indicate that the G helix likely undergoes changes in the early M and ML states, due to its direct bonding to the retinal chromophore and the role of the SB counterion residue Asp212. These changes disappear during the M[.-~M[,, transition. However, the F helix is relatively inert to the photoreactions in the first half of the bR photocycle.

来自X射线、中子衍射和电子显微镜的证据 表明F螺旋在bR光循环期间是倾斜的， SB区进入质子和bR的G-螺旋 在质子运输过程中发生了显著的结构变化。 巧合的是，bR中的21个缬氨酸残基中有10个位于这两个 螺旋，F中7个（瓦尔167、173、177、179、180、187和188），G中3个 （Va 1210，213和217）。 根据亨德森的bR结构模型，681 有两个直接键合的缬氨酸对（Vall 79和VaI 180，VaI 187 和VaI 188）在富含缬氨酸的F-螺旋中。 在每一个， 羰基在空间上接近（-3AO）。 之间的距离 F中的缬氨酸对VaI 177和VaI 179，A Va 1210和V11213，以及Va 1213 G中的Va 1217和Va 1217的分子量约为4.5A-5。 光适应（LA）状态和M之间的差谱。 [1- 13 CVal]、[15 N-Prol-bR]的光中间体显示， 21个缬氨酸残基中至少两个的位移向高场移动 至少3.5 ppm，这表明接近 这些缬氨酸残基。 这 意味着这些缬氨酸残基是 在bR光循环期间活跃。 选择性观察缬氨酸49， 69和199通过异核偶极再偶联（REDOR）消除了 三个缬氨酸残基，这不是在 无论是F或G螺旋，负责观察到的变化， NMR差谱。 因此， 研究这些变化是否发生在F和 LA-+M中的G螺旋。 过渡 同向偶极再耦合 （MELODRAMA）实验在短的混合时间下进行， 为了仅使相距约3 A的两个缬氨酸对重新偶联， 更长的混合时间以关联F和G中的三个缬氨酸对 螺旋，进一步分离。 结果表明，G 螺旋可能在早期的M和ML状态下发生变化，这是由于 它与视网膜发色团的直接结合以及SB的作用 互补残基Asp 212。 这些变化在 M[.-~ M[，transition. 然而，F螺旋对蛋白质是相对惰性的。 在bR光循环的前半部分的光反应。