Mechanism of energy transduction by bacteriorhodopsin

细菌视紫红质的能量转换机制

• 批准号: 8344746
• 负责人: richard w hendler
• 金额: $ 0.78万
• 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/8344746
• 关键词: Animals; Bacteriorhodopsins; Behavior; Caliber; Complex; Computer software; Custom; Data; Detergents; Development; Electron Transport; Employee Strikes; Environment; Event; Fiber Optics; Goals; Image; In Situ; Kinetics; Laboratories; Laboratory Research; Lasers; Light; Light Microscope; Manufacturer Name; Masks; Membrane; Membrane Proteins; Microscope; Microscopic; Modeling; Modification; Molecular; Monitor; Optics; Organism; Oxygen; Paper; Photons; Physiologic pulse; Problem Solving; Procedures; Proteins; Proton Pump; Protons; Purple Membrane; Research Personnel; Respiratory Chain; Sampling; Schedule; Solutions; Spectrometry; Spectrum Analysis; System; Testing; Time; United States National Institutes of Health; Water; Width; Work; X ray diffraction analysis; X-Ray Diffraction; air sampling; base; charge coupled device camera; cytochrome c oxidase; in vivo; infrared spectroscopy; instrument; instrumentation; lens; light intensity; meetings; operation; programs; quantum; research study; response; skills; time interval; transmission process; voltage

Summary: A.) Combined IR and optical spectrometry This part of the project has been completed and the first paper is scheduled for the September issue of Applied Spectroscopy. Its unique feature is using linear algebraic procedures, developed in my laboratory, to obtain for the first time, absolute visible and IR spectra for all intermediates in the bacteriorhodopsin (BR) photocycle. The ability to isolate spectra for each intermediate is based on the parallel cycle model supported in my laboratory, rather than the alternative single cycle model accepted by a large group of research laboratories. With these isolated spectra, we were able to obtain the transitional difference spectra between consecutive intermediates, rather than the difference spectra between each intermediate and the ground state, as done previously. We were also able to separate (for the first time), the spectra and kinetics of two very similar forms of the M intermediate. Using these new sequential spectra, we were able to describe new features and functions of the BR photocycle. B.) Development of instrumentation and procedures for comparing visible kinetics of the BR photocycle in membrane protein crystals to that of in situ tiny membrane fragments. Crystals of functional proteins are widely perceived and used as models for how proteins act in vivo. This is a particularly tenuous assumption for membrane proteins because they are most often separated from the membrane using detergents. We think it is essential to establish how near or far such crystals mimic the activities of the in situ protein. Towards this end, we have been trying to develop instrumentation and software to provide an answer to this question. The basis of our approach is to apply the new combined visible and IR spectroscopic approach described above, to crystals of BR and to similarly small samples of BR in its native purple membrane (PM) environment. This has proven to be a challenge that we are still trying to meet. In the studies described in Part A, the sample was in a circle of 1 cm diameter. For crystals, the microscopic sample will be in a circle of 50 or 100 microns. For any given intensity of monitoring light passing through a 50 micron orifice there will be only 1/40000th as much light and through a 100 micron opening, 1/10000th . We have been pursuing two separate approaches to obtain visible kinetic spectra; one based on the unique spectrophotometer built for me at NIH several years ago, and the other using a Princeton Instruments CCD camera/spectrometer with and without an image intensifier. The kinetic experiment must be monitored with a light intensity not high enough to initiate any turnover of the BR photocycle. A single turnover of the cycle is then initiated by an intense 5 ns laser pulse. Using both fiber optics and lenses, we were able to conduct light from the microscope to the NIH-built spectrometer. These efforts to obtain visible kinetics with the NIH spectrometer have not succeeded. The intensity of monitoring light needed to record sufficient photon counts was above the threshold for initiating turnover. The commercial CCD/spectrometer has presented a number of other problems; 1.) Its response to increasing light intensity is linear only up to about 1/2 of its range. 2.) Using constantly spaced time intervals with a sample of air or water, the count level at each time point unexpectedly rises from about mid-point in the schedule, instead of remaining constant. 3.) When using kinetics that involve different time intervals for collecting data, the recorded photon counts are not linearly related to the dwell times of the different intervals. In principle, the image intensifier (ii) that we have should solve these problems by acting as a shutter or gate. In this mode, the ii placed between the spectrometer and CCD camera blocks all light except that which is programmed for a constant scheduled pulse width of 1-2 microsec. In addition, it provides a very significant amplification for the photon level it receives. The ii works as follows: Photons strike a photocathode that produces a current that is greatly amplified as it passes through a microchannel plate and the enhanced current strikes a phosphorescent screen that emits photons for the CCD to record. The problem is that if the enhanced current is too high, it could permanently damage the ii. To avoid this, a control circuit automatically cuts in (without warning) to decrease the gain. The resultant changes in photon counts totally obscure the changes due to the turnover of BR. After much discussion with the manufacturer, changes were made in the circuitry that gives us more room to use the pulse mode of operation safely. This does work, and the problems listed above for the CCD disappeared. But, the ii has introduced a new serious problem. The quantum efficiency (qe) of its phosphor is very low in the range near 412 nm where one of the most important intermediates of the photocycle occurs. The qe is drastically higher at the higher wavelengths where the other intermediates are found. With the 12-bit A/D converter, our maximum count level must be lower than 65536 counts to avoid saturation. This limits us to too few counts for following the 412 nm intermediate. There is one solution that should overcome this problem. We need a custom filter made just for the emission spectrum of the ii phosphor such that its transmission will compensate at all wavelengths to provide a near constant level of transmittance counts. With this, we can expect to increase the approximately 1500 counts we now obtain at 412 nm to about 50000 counts. I tested this idea using a variety of filters and other means to modify the photon emission spectrum and was able to raise the count level at 412 nm to 5000 counts. With the custom filter, we expect a further 10-fold enhancement in the count level. C.) At the CARB facility of NIST. Development of instrumentation for studying IR kinetics of the BR photocycle in single membrane/protein crystals and tiny membrane fragments. The newly acquired Bruker IR spectrometer and microscope are working and we can obtain IR kinetics using the 100 micron window. Our crystallographer has acquired the skill to make BR crystals. Brucker is making modifications that should allow us to work with a 50 micron mask. Once we have solved the existing problems with the visible system, we will move it to NIST for the combined IR/visible kinetic studies on crystals and membrane fragments.

摘要：A。）组合IR和光谱法 该项目的这一部分已经完成，第一篇论文安排在9月的应用光谱法上。它的独特特征是使用我的实验室中开发的线性代数程序，以便首次获得细菌光蛋白（BR）光周期中所有中间体的绝对可见和红外光谱。每个中间体分离光谱的能力是基于我实验室支持的平行循环模型，而不是大量研究实验室接受的替代单周期模型。使用这些孤立的光谱，我们能够获得连续中间体之间的过渡差异光谱，而不是以前所做的那样，而不是每个中间状态和基态之间的差异光谱。我们还能够分开（这是第一次），这两种非常相似的M中间形式的光谱和动力学。使用这些新的顺序光谱，我们能够描述BR光循环的新功能和功能。 B.）开发仪器和程序，用于比较膜蛋白晶体中BR光循环的可见动力学与原位微小膜片段的动力学。 功能蛋白的晶体被广泛感知并用作蛋白质在体内作用的模型。对于膜蛋白来说，这是一个特别脆弱的假设，因为它们通常使用洗涤剂与膜分开。我们认为必须确定此类晶体模仿原位蛋白的活性是至关重要的。为此，我们一直在努力开发仪器和软件，以便为这个问题提供答案。我们方法的基础是将上述新的合并可见和红外光谱方法应用于BR的晶体，并将其在其天然紫色膜（PM）环境中类似的BR样品中。事实证明，这是我们仍在努力应对的挑战。 在A部分中描述的研究中，样品的直径为1 cm。对于晶体，微观样品将以50或100微米为单位。对于通过50微米孔口的任何给定的监视光强度，只有1/40000的光线和100微米的开口，1/10000。我们一直在采用两种单独的方法来获得可见的动力学光谱。一个基于几年前在NIH上为我建造的独特分光光度计，另一个是使用普林斯顿仪器CCD摄像头/光谱仪，带有和没有图像增强器的光谱仪。动力学实验必须以未足够高的光强度来监测以启动BR光循环的任何营业额。然后，通过强烈的5 ns激光脉冲开始循环的单个周期。 使用光纤和镜头，我们能够从显微镜到NIH建造的光谱仪进行光。通过NIH光谱仪获得可见动力学的这些努力尚未成功。 记录足够的光子计数所需的监测光强度高于启动营业额的阈值。 商业CCD/光谱仪提出了许多其他问题。 1.）其对增强光强度的响应仅是线性的，最大约为其范围的1/2。 2.）使用空气或水样品使用不断间隔的时间间隔，每个时间点的计数水平出乎意料地从时间表中的大约中点上升，而不是保持恒定。 3.）当使用涉及不同时间间隔来收集数据的动力学时，记录的光子计数与不同间隔的停留时间无线性相关。 原则上，我们应该通过充当快门或门来解决这些问题的图像增强器（II）。在此模式下，将II放置在光谱仪和CCD摄像机之间的II都阻止了所有光，除了以1-2 microSec的恒定计划脉冲宽度进行编程。此外，它为收到的光子水平提供了非常明显的扩增。 II的作用如下：光子撞击了一个光电阴道，该光电阴道会产生一个电流，该电流通过微通道板时大大放大，并且增强的电流击中了磷光屏幕，该磷光屏幕发射光子以记录CCD。问题是，如果增强电流太高，它可能会永久损坏II。为了避免这种情况，控制电路会自动切入（无警告）以降低增益。由于BR的营业额而导致的变化，因此光子计数的结果变化完全掩盖了。在与制造商进行了大量讨论之后，在电路中进行了更改，这使我们更有空间安全地使用脉冲模式。这确实有效，并且CCD上面列出的问题消失了。但是，II引入了一个新的严重问题。在412 nm附近，其磷光体的量子效率（QE）非常低，其中最重要的中间体之一发生。在发现其他中间体的较高波长下，量化宽度的量很大。使用12位A/D转换器，我们的最大计数水平必须低于65536计数，以避免饱和。这限制了我们遵循412 nm中间体的计数。有一种解决方案应该克服这个问题。我们需要仅针对II磷光器的发射光谱制成的自定义过滤器，以便其传输将在所有波长下进行补偿，以提供接近恒定的透射率计数。这样，我们可以期望将现在412 nm的大约1500个计数增加到约50000个计数。我使用多种过滤器和其他方法来修改光子发射光谱，并能够将计数水平提高到412 nm至5000个计数。使用自定义过滤器，我们预计计数水平会进一步增强10倍。 C.）在NIST的Carb设施中。在单膜/蛋白质晶体和微小的膜片段中研究BR光循环的IR动力学的仪器开发。 新获得的布鲁克红外光谱仪和显微镜正在工作，我们可以使用100微米窗口获得IR动力学。我们的晶体学家已经获得了制造BR晶体的技能。布鲁克正在进行修改，以使我们能够使用50微米面膜。一旦我们解决了可见系统的现有问题，我们将其将其移至NIST，以进行IR/可见的晶体和膜片段的合并动力学研究。