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首页> 外文期刊>Biochemistry >Structure of the interhelical loops and carboxyl terminus of bacteriorhodopsin by X-ray diffraction using site-directed heavy-atom labeling
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Structure of the interhelical loops and carboxyl terminus of bacteriorhodopsin by X-ray diffraction using site-directed heavy-atom labeling

机译：细菌视紫红质的螺旋间环和羧基末端的结构X射线衍射的定点重原子标记

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The positions of single amino acids in the interhelical loop regions and the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild-type bR does not contain any cysteines, appropriate cysteine mutants were produced with a unique sulfhydryl group at specific positions. These sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V101C, V130C, A160C, and G231C were derivatized with labeling stoichiometries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respectively (Hg per bR). No incorporation was observed with wild-type bR under the same conditions. All mutants and heavy-atom derivatives were fully active as judged by the kinetics of the photocycle and of the proton release and uptake. Moreover, the unit cell dimensions of the two-dimensional P-3 lattice were unchanged by the mutations and the derivatization. This allowed the position of the mercury atoms, projected onto the plane of the membrane, to be calculated from the intensity differences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. These native conditions of temperature, humidity, and solvent are expected to preserve the structure of the surface-exposed loops. Sharp maxima corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localized difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more disordered and mobile part of the structure. [References: 32]
机译：通过使用定点重原子标记的X射线衍射研究了细菌视紫红质（bR）的螺旋间环区和C末端尾巴中单个氨基酸的位置。由于野生型bR不包含任何半胱氨酸，因此产生了合适的半胱氨酸突变体，在特定位置带有独特的巯基。然后使用巯基特异性试剂对氯mercuribenzoate（p-CMB）用汞标记这些位点。半胱氨酸突变体D96A / V101C，V130C，A160C和G231C的标记化学计量比分别为0.93 +/- 5％，0.85 +/- 5％，0.79 +/- 7％和0.77 +/- 8％（ Hg / bR）。在相同条件下未观察到野生型bR的掺入。根据光循环动力学以及质子释放和吸收的动力学判断，所有突变体和重原子衍生物均具有充分的活性。此外，二维P-3晶格的晶胞尺寸通过突变和衍生化而保持不变。这样就可以使用傅立叶差分法根据标记样品和未标记样品之间X射线衍射图的强度差来计算投影到膜平面上的汞原子的位置。室温下，在不使用脱水溶剂的情况下，在100％相对湿度下从取向紫色膜薄膜收集X射线衍射数据。这些自然的温度，湿度和溶剂条件有望保留暴露于表面的环的结构。在D96A / V101C和V130C的差异密度图中发现了对应于单个汞原子的尖锐最大值。残基101和130分别处于连接螺旋C / D和D / E的短环中。没有发现A160C和G231C的局部差异密度。残基160在连接螺旋E和F的较长环中，而残基231在C-末端尾巴中。残基160和231显然在结构的更无序和可移动的部分中。 [参考：32]

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