Mechanism of energy transduction by bacteriorhodopsin

细菌视紫红质的能量转换机制

• 批准号: 6966864
• 负责人: richard w hendler
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966864
• 关键词: Halobacteriaceae; acidity /alkalinity; bacterial proteins; bacteriorhodopsins; bioenergetics; hydrogen ions; lipid bilayer membrane; membrane channels; photoactivation; photonics; photosynthetic bacteria; protein structure function; squalene; temperature

The purple membrane (PM) from H. Salinarium contains a single, well defined, 26000 D protein (bacteriorhodopsin (bR)) in intimate association with 10 lipid molecules. The monomer is part of a trimer arranged in a symmetric, hexagonal, two dimensional crystal. PM performs the same function as a respiratory chain in its ability to transduce an input energy into a electrochemical proton gradient used to form ATP. Our laboratory has shown that specific lipids of the membrane control the conformational flexibility of bR alpha-helices and the photocycle itself. Specifically, squalene appears to mitigate a repulsive interaction of polar lipids with aspartates in the cytoplasmic loop region to lesson a contortional strain which decreases the kinetics of the photocycle. Our laboratory has challenged the prevailing view that the photocycle is an homogeneous series of reversible linear conversions of intermediates (RHM) represented as BR <-> K <-> L <-> M1 <-> M2 <-> N <-> O <-> BR. In this view, the M1 -> M2 conversion represents an essential gate for proton transport where the retinal in a Schiff base with lys-216 shifts its orientation from a cytoplasmic channel to an outside channel. Extensive X-ray diffraction evidence has been produced to support this view. However, our laboratory has shown that the bR photocycle, at pH 7 and 20 degrees C, is better represented by separate unidirectional parallel cycles (UPM) , specifically, BR -> K -> L -> M1 -> N -> O -> BR, and BR ->K -> L -> M2 -> BR. If the M1 and M2 are in separate cycles, the proton gate model can not be true. This past year, we have shown that the UPM fits experimental data both from our laboratory and others better than does the RHM. We also have established the universality of the UPM in its ability to deconvolute raw data collected under nine conditions of varied temperature and pH. In this survey, we have found two additional photocycles that become prominent at different pH?s and T' s: BR -> K -> L -> M -> O -> BR, and BR -> K -> L -> M -> N -> BR. If one plots the the locations of cycles on a grid of pH in one dimension and T in the other, a phase-like diagram is seen in which different combinations of the four basic cycles are found in common areas of pH and T. This contradicts the current view that only a single RHM exists and that its kinetics as a function of T observe the Arrhenius equation. Because the RHM and UPM are entirely distinct, the fitting of UPM to all of these different data sets strongly supports the validity of UPM unless, the UPM can unexplainably also fit data generated by a RHM. To test this possibility we synthesized data based on RHM and demonstrated that they could not be fit by a UPM. Based on our ability to extract the absolute spectrum for each intermediate from raw kinetic data, we have started a collaboration with Aleksandr Smirnov to try to obtain structures for each intermediate in the photocycle using time-resolved X-Ray diffraction data. With a simultaneous kinetic study of the magnitudes of proton current and voltage formation during each step of the photocycle we hope to learn much more about the proton-pumping process and the ability of the membrane to control it.

来自H. Salinarium的紫色膜（PM）包含一个与10个脂质分子密切关联的单个，定义良好的26000 d蛋白（细菌霍顿（BR））。该单体是排列在对称，六角二维晶体中的三聚体的一部分。 PM在将输入能量转换为用于形成ATP的电化学质子梯度的能力方面具有与呼吸链相同的功能。我们的实验室表明，膜的特定脂质控制BRα-螺旋和光循环本身的构象柔韧性。具体而言，沙列烯似乎减轻了极性脂质与细胞质环区域中天冬氨酸的排斥相互作用，从而降低了光圈动力学的扭曲菌株。我们的实验室挑战了普遍的观点，即光循环是一系列均匀的中间体的可逆线性转换（RHM） br <-> k <-> l <-> m1 <-> m2 <-> n <-> o <-> br。在此观点中，M1-> M2转换代表了质子转运的重要门，其中带有LYS -216的Schiff底座中的视网膜将其方向从细胞质通道转移到外部通道。已经产生了广泛的X射线衍射证据来支持这一观点。但是，我们的实验室表明，在pH 7和20摄氏度下的BR光循环更好地由单独的单向平行循环（UPM）表示，具体来说，BR-> k-> k-> k-> l-> l-> m1-> n-> n-> n-> o-> br-> br-> k-> k-> k-> k-> l-> l-> m2-> m2-> br。如果M1和M2在单独的周期中，则质子栅极模型是不正确的。在过去的一年中，我们已经证明，UPM比RHM更适合我们实验室的实验数据。我们还建立了UPM的普遍性，其能够在九种温度和pH的条件下取消收集的原始数据。在这项调查中，我们发现了另外两个在不同的pH？s and t's：br-> k-> l-> m-> m-> m-> o-> br和br-> k-> k-> l-> m-> m-> n-> n-> br。如果一个人在一个维度上绘制了一个pH的位置，另一个维度绘制了pH的网格的位置，则可以看到一个类似相图的图，其中在pH和T的公共区域中发现了四个基本周期的不同组合。这与当前的观点相矛盾，该观点只有单个RHM存在，并且它的动力学与therhenius公平的动力相吻合。由于RHM和UPM完全不同，因此UPM与所有这些不同数据集的拟合强烈支持UPM的有效性，除非UPM无法解释，也可以符合RHM生成的数据。为了测试这种可能性，我们基于RHM合成数据，并证明它们不能被UPM拟合。基于我们从原始动力学数据中提取每个中间体的绝对频谱的能力，我们已经开始与Aleksandr Smirnov合作，以尝试使用时间分辨的X射线衍射数据来获取光循环中每个中间体的结构。通过同时研究光循环每一步中质子电流和电压形成的幅度的动力学研究，我们希望更多地了解有关质子泵送过程的更多信息以及膜控制质子的能力。