EFFICIENCIES OF ENERGY TRANSDUCTION BY BACTERIORHODOPSIN

细菌视紫红质的能量转换效率

基本信息

批准号：
6432640
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
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

In last year's report, we described the lack of correlation between the kinetics of the bacteriorhodopsin (BR) photocycle measured by optical spectroscopy and by voltage generation across the energy-transducing membrane. We considered and evaluated two different explanations. 1.) Thermodynamic back-pressure directly decreased kinetic constants in the photocycle transitions. 2.) The apparent difference was explained by slow major electrogenic events that showed negligible optical changes. In this case, the effects of adding uncouplers would be due to their lowering of the electrical resistance (R) of the membrane and consequently the RC ( C for capacitance) constant leading to faster electrical response times. Our results, at the time, favored explanation 1. Because, of the importance of establishing the true explanation for the phenomenon, we repeated and extended the controls that had been performed earlier. The new data confirmed our earlier conclusions and the work was submitted for publication in The European Journal of Biochemistry and accepted. In order to achieve the original goal of correlating the kinetics of voltage generation with the optical kinetics of photocycle turnover to identify the energy-transducing steps, we turned to a new approach. The system we used to follow the kinetics of voltage generation consists of small samples of fixed membranes in a closed vesicular conformation. The voltage that builds causes the slowdown in kinetics. We have started to construct a new system which uses open membrane fragments electrically oriented in a gel. No standing voltage is formed, but with electrodes, one can measure the kinetics of current (i.e. pumped protons) movements. An additional advantage of this system is that both the optical and electrical responses can be measured on the same sample. Combining this approach with our new discovery of a specific kinetic model which describes the BR-photocycle (described in Z01 HL00401-35) could lead to the identification of the energy-transducing steps in the photocycle. The kinetic model involves two separate, but parallel photocycles, one with five steps, and the other with three. We believe that the 5-step cycle may be energy-transducing and that the 3-step cycle, energy-dissipating. In previous reports, we described experimental data which implicated the importance of a membrane squalene-phosphatidyl glycerophosphate interaction with a BR-peripheral aspartic acid residue for the energy-transducing function of the photocycle. To test this idea, we arranged with Dr. Martin Engelhard in Germany to prepare 15-C-aspartic acid-labeled-BR. In collaboration with Dr. Robert Tycko of NIDDK, we examined the solid state NMR signals from this preparation and compared them with a Triton X-100-treated preparation, which is known to destroy the suspected energy-transducing part of the photocycle. The results indicated a subtle perturbation in signals that have not been assigned to any of the internal aspartic acid residues. Therefore, these may be due to the peripheral residues. We plan to repeat these experiments and to find if reconstitution of the Triton-damaged preparation with extracted membrane lipids, under conditions known to restore native behavior to the membranes, restores the native pattern for signals from the labeled aspartate residues.

在去年的报告中，我们描述了通过光谱法测定的细菌蛋白蛋白（BR）光循环的动力学之间的相关性，并且通过跨能透射膜进行了电压产生。我们考虑并评估了两种不同的解释。 1.）热力学后压在光循环转变中直接降低动力学常数。 2.）明显的差异是通过缓慢的重大电源事件来解释的，这些事件显示出可忽略不计的光学变化。在这种情况下，添加解耦合器的效果将是由于它们降低了膜的电阻（R），因此RC（用于电容的C）常数导致电响应时间更快。当时我们的结果赞成解释1。因为，建立对现象的真实解释的重要性，我们重复并扩展了较早执行的控制。新数据证实了我们的早期结论，这项工作已在《欧洲生物化学杂志》上发表并接受。为了实现将电压产生动力学与光周期转换的光学动力学相关联的最初目标，以识别能量传递步骤，我们转向了一种新方法。我们过去遵循电压产生动力学的系统由封闭的囊泡构象中的固定膜的小样品组成。构建的电压导致动力学的放缓。我们已经开始构建一个新的系统，该系统使用凝胶中电向的开放膜片段。没有形成站立电压，但是使用电极，可以测量电流（即泵送质子）运动的动力学。该系统的另一个优点是，可以在同一样品上测量光学响应和电气响应。将这种方法与我们新发现的特定动力学模型相结合，该模型描述了BR-Photocycle（在Z01 HL00401-35中进行了描述）可能会导致鉴定光周期中能量传递步骤。动力学模型涉及两个单独但平行的光循环，一个具有五个步骤，另一个带有三个步骤。我们认为，五步周期可能是能量传递的，并且三步周期会消耗能量。在先前的报道中，我们描述了实验数据，这暗示了膜 - 沙列烯基磷脂酰磷酸磷酸磷酸磷酸磷酸的重要性与BR-外膜天冬氨酸残基在光循环功能中的重要性。为了测试这个想法，我们与德国的马丁·恩格哈德（Martin Engelhard）博士安排，准备了15-C-天冬氨酸标记的BR。与NIDDK的Robert Tycko博士合作，我们检查了此准备中的固态NMR信号，并将其与Triton X-100处理的制剂进行了比较，众所周知，该制剂可破坏光循环的可疑能量传递部分。结果表明，尚未分配给任何内部天冬氨酸残基的信号中的微妙扰动。因此，这些可能是由于周围残基引起的。我们计划重复这些实验，并发现是否在已知恢复膜的天然行为的条件下重新建立了Triton损坏的制剂，还可以恢复来自标记为Aspartate残基的信号的天然模式。