Mechanism of energy transduction by bacteriorhodopsin

细菌视紫红质的能量转换机制

• 批准号: 7968970
• 负责人: richard w hendler
• 金额: $ 0.68万
• 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/7968970
• 关键词: Achievement; Address; Amino Acids; Animals; Area; Bacteriorhodopsins; Behavior; Binding; Biochemical; Biotechnology; Cells; Charge; Collaborations; Complex; Consultations; Crystallization; Data; Defect; Detergents; Devices; Electron Transport; Electronics; Electrons; Engineering; Environment; Equipment; Event; Exposure to; Fiber; Goals; Growth; Hand; Head; Height; Hydrogen Bonding; Image; Joints; Kinetics; Laboratories; Lasers; Light; Lipids; Location; Manufacturer Name; Manuscripts; Masks; Measures; Membrane; Membrane Lipids; Membrane Proteins; Microscope; Molecular; Molecular Structure; Monitor; Movement; National Heart, Lung, and Blood Institute; National Institute of Biomedical Imaging and Bioengineering; Noise; Ocular orbit; Optics; Organism; Oxygen; Pattern; Photons; Physiologic pulse; Positioning Attribute; Preparation; Problem Solving; Procedures; Process; Proteins; Proton Pump; Protons; Purple Membrane; Research; Research Personnel; Respiratory Chain; Roentgen Rays; Sample Size; Sampling; Schedule; Science; Side; Signal Transduction; Spectrometry; System; Testing; Time; United States National Institutes of Health; Universities; Visible Radiation; Work; X ray diffraction analysis; X-Ray Diffraction; charge coupled device camera; chromophore; cytochrome c oxidase; data acquisition; detector; experience; infrared spectroscopy; instrument; instrumentation; interest; protein structure; stem; vibration; voltage

1. Combined IR and optical spectrometry. During the past year we were successful in combining optical and IR spectroscopies, using the same sample of BR, in order to isolate absolute spectra for each intermediate in the BR photocycle. By subtracting the absolute IR spectrum for each intermediate in successive steps of the photocycle, we have obtained information on structural changes of the protein and proton-binding to the amino acids as the proton is transported across the membrane. IR spectra are much more complex than optical spectra and contain much more information. Optical spectra are produced by specific chromophores in which electrons are elevated to higher energy orbits. The spectral features are simple, broad, and Gaussian-like. IR spectra emanate from atomic vibrations from every atomic bond. IR difference spectra between two sequential intermediates contain a great number of sharp positive and negative features, due, for example, to changes in hydrogen-bonds, protein structure or environment, and proton-binding to particular amino acids. To more fully understand the many changes we see in the difference IR spectra we have obtained, we have invited an expert in the interpretation of IR spectral features from proteins to join us as a collaborator. He is Mark Braiman from Syracuse University. His earlier IR studies of the BR Photocycle helped establish the basic understanding of some of the steps of proton movements across the membrane. He accepted our offer and we will send him a manuscript of the completed work described here. 2. Studies using single crystals of membrane protein (BR) This is a joint collaborative project involving NHLBI, NIBIB, and CIT from NIH and the Biochemical Science Division of NIST. Stemming largely from my collaboration with Dr. Meuse described above, he has been transferred from the Biospectroscopy Group to the Macromolecular Structure and Function Group. The goal of this project is to develop instrumentation and approaches to study and compare the functionality of a pure crystal of membrane protein to that of the same protein in its native membrane. Functionality will be measured by combined optical and IR spectral kinetics. No instrument capable of performing such studies has been described previously. The initial optical studies are being conducted at NIH in the laboratory of Paul D. Smith at NIBIB and the IR studies at the NIST location at CARB (Center for Advanced Studies in Biotechnology). As soon as the optical system is ready, it will be moved to CARB for integration with the IR system. At NIH, NIBIB has acquired a charge-coupled device (CCD) camera with an attached spectrograph. The CCD has a photon detector array that contains 1048 rows of 512 pixels each. Each row can record a 512 wavelength spectrum at up to 1048 different time points. The pixel size is 16 microns (u). To obtain kinetic data, light must be focused on the bottom row and the row shifts below a mask after it has been exposed for a set time-increment to allow the next row to be exposed. To increase the size of the signal, the window size (WS) can be increased to more than a single row at a time. We have been using a WS of 2, thus reducing the number of time points to 524. The device allows only fixed time-increments and no repeat data acquisitions to increase the signal to noise ratio. John Kakareka (CIT) has built an electronic timing device that allows the use of staggered time-increments to cover the whole range of interest for the BR photocycle. We have built a special sample cell that accepts a sample size of less than 2 ul. A 600 u fiber brings the monitoring light to the sample and a 200 u fiber about 400 u away conducts the transmitted light to the spectrograph slit. A 600 u fiber at right angles to the 2 fibers described above transmits the laser pulse to the sample. The 200 u circle of transmitted light is masked on the sides by the spectrometer slit and vertically confined by a knife-edge mask, controlled in position by a vernier device. This is necessary to minimize the spill of light outside of the 32 u height of 2 rows. Using this setup, we are able to observe photocycle turnover. There are a number of technical problems we must solve before the device will be ready for moving to CARB. 1.) We must integrate a trigger-initiated pulse laser into the system for synchronizing the starts of multiple photocycles to allow averaging and achievement of high signal to noise ratios. 2.) Because, the detector array integrates photons over the whole time- increment period, there is no precise point of time that can be assigned to the amount of signal acquired. This is a very big problem when 4 or 5 different time increments are used during a complete photocycle turnover. We plan to solve this problem with an image-intensifier that has been ordered. A very low level of monitoring light will be used and the intensifier maintained in a closed state until the end of each time-increment scheduled. At that time a very sharp electrical signal will activate the intensifier for a period of ns to acquire data at precise points in time. 3.) The CCD has some defects that must be addressed by consultation with the manufacturer. In particular, there are drifts in signal level over time with H2O as the sample. The time course should be perfectly stable at all wavelengths. This may be as simple as scheduling cleans at appropriate times to remove static charge build-ups. On the other hand it might require discussions with the engineers and designer of the instrument. At NIST (CARB) a new IR spectrometer and an accompanying dedicated microscope were ordered and received. This microscope has its own infrared detector and optics for focusing both IR and visible light. It is capable of using either a single crystal of membrane protein or a tiny fragment of the purple membrane (PM) containing BR. The first task at CARB will be to set up and become familiar with this new equipment. The Macromolecular Structure and Function Group has several X-ray crystallographers on the staff. The head of the group is interested in moving in to the area of membrane proteins and has assigned an experienced crystallographer, Jane Ladner, to work with us on this project. The plan is to start with procedures that have led to the growth of BR crystals in order to test all of our new equipment and to compare the functionality of the crystals with that of BR in its native environment. Because, these crystals were exposed to detergent, and because their lipid environment is not the same as the lipids in PM I know from my own research that the kinetic behavior will not be the same. We then plan to modify the crystallization procedures to minimize exposure to detergent and to either introduce native PM lipids or to replace the foreign lipids entirely with PM lipids. For each new preparation, we will compare the crystals to PM. With the best preparations, we will isolate absolute optical and IR spectra. New directions: With the most native-like crystals, we plan to obtain time-resolved X-ray diffraction data. Using the same procedures which led to our obtaining isolated, absolute IR spectra, we should be able to obtained isolated diffraction patterns for each intermediate in the photocycle. If successful, this should show the position of each atom in the protein as it transports a proton across the membrane to form an electrochemical potential. This information should add significantly to the understanding of the process of energy transduction by proton pumps, such as cytochrome oxidase.

1。组合IR和光谱法。 在过去的一年中，我们成功地使用了相同的BR样品将光学光谱和IR光谱镜结合在一起，以分离BR光循环中每个中间体的绝对光谱。通过在光循环的连续步骤中减去每个中间体的绝对红外光谱，我们在质子跨膜上运输时，已经获得了有关蛋白质和质子结合的结构变化的信息。红外光谱比光谱要复杂得多，并且包含更多信息。光谱是由特定的发色团产生的，其中电子升高到较高的能轨道。光谱特征简单，宽，高斯般。 IR光谱来自每个原子键的原子振动。两个顺序中间体之间的IR差异光谱包含大量尖锐的正和负特征，例如由于氢键的变化，蛋白质结构或环境的变化以及对特定氨基酸的质子结合。为了更充分地了解我们在获得的差异光谱中看到的许多变化，我们邀请了专家在解释蛋白质的IR光谱特征的解释中加入我们作为合作者。他是锡拉丘兹大学的马克·布雷曼（Mark Braiman）。他先前对BR光循环的IR研究有助于建立对整个膜运动的某些步骤的基本理解。他接受了我们的提议，我们将向他发送有关此处描述的完成工作的手稿。 2。使用膜蛋白的单晶（BR）的研究 这是一个联合合作项目，涉及NHLBI，NIBIB和NIH的CIT和NIST的生化科学部。在很大程度上源于我与上述Meuse博士的合作，他已从生物光谱群体转移到大分子结构和功能组。该项目的目的是开发仪器和方法，以研究和比较膜蛋白的纯晶体与天然膜中同一蛋白质的晶体功能。功能将通过光学光谱动力学组合来测量。 先前尚无能力进行此类研究的仪器。最初的光学研究是在NIH的NIH进行的，在NIBIB的Paul D. Smith实验室和Carb的NIST位置的IR研究（生物技术高级研究中心）。 一旦准备好光学系统，它将移至碳水化合物以与IR系统集成。 在NIH，尼比布（Nibib）购买了带有光谱仪的电荷耦合设备（CCD）摄像头。 CCD的光子检测器阵列包含1048行，每个行分别为512个像素。每一行最多可以在1048个不同的时间点记录512波长光谱。像素大小为16微米（u）。为了获得动力学数据，必须将光集中在底部行上，并且在掩模以下掩模后，行移动以进行设定的时间提示，以使下一行暴露出来。为了增加信号的大小，窗口大小（WS）一次可以增加到一排超过一行。我们一直使用2个WS，从而将时间点的数量降低到524。该设备仅允许固定的时间提示，而无重复数据采集来增加信号与噪声比。约翰·卡卡雷卡（John Kakareka）（CIT）已经建立了一个电子时序设备，该设备允许使用交错的时间增加来覆盖BR光循环的整个兴趣范围。我们已经建立了一个特殊的样品单元，该样品单元的样本量小于2 ul。 600 U纤维将监视光带到样品中，200 U纤维约400 U远可将传输的灯传输到光谱仪的缝隙。与上述2个纤维成直角的600 U纤维将激光脉冲传输到样品。 200 U圆的发射光圆通过光谱仪缝在侧面掩盖，并垂直被刀口遮罩限制，并由Vernier设备控制在位。 这是最大程度地减少32 U高度2行之外的光的溢出所必需的。使用此设置，我们能够观察光周期的周转。我们必须解决许多技术问题，然后才能准备好搬运碳水化合物。 1.）我们必须将触发引发的脉冲激光器集成到系统中，以同步多个光循环的开始，以平均和实现高信号与噪声比。 2.）因为，检测器阵列在整个时间增量期间都集成了光子，所以没有精确的时间点可以分配给获得的信号量。当完整的光循环周转期间使用4或5个不同的时间增量时，这是一个非常大的问题。我们计划使用已订购的图像增强器来解决此问题。将使用非常低的监视灯，并保持封闭状态的增强剂，直到每个时间提议结束为止。当时，非常尖锐的电信号将在NS期间激活增强器，以便在精确的时间点获取数据。 3.）CCD有一些缺陷，必须通过与制造商的磋商来解决。特别是，随着时间的流逝，H2O作为样品的信号级别存在漂移。时间过程在所有波长上都应完全稳定。这可能就像在适当的时间安排清洁以删除静态电荷积累一样简单。另一方面，它可能需要与乐器的工程师和设计师进行讨论。 在NIST（CARB），订购并接收了一个新的红外光谱仪和随附的专用显微镜。该显微镜具有自己的红外探测器和光学元件，用于聚焦IR和可见光。它能够使用膜蛋白的单个晶体或含有br的紫色膜（PM）的微小片段。卡布的第一个任务是设置并熟悉这种新设备。大分子结构和功能组有几位X射线晶体学者。该小组的负责人有兴趣进入膜蛋白的区域，并分配了一位经验丰富的晶体学家Jane Ladner，与我们合作。 该计划是从导致BR晶体生长的过程开始，以测试我们所有的新设备，并将晶体的功能与BR在本机环境中的功能进行比较。因为，这些晶体暴露于洗涤剂，并且由于它们的脂质环境与PM中的脂质不同，所以我从自己的研究中知道，动力学行为不会相同。然后，我们计划修改结晶程序，以最大程度地减少对洗涤剂的接触，并引入天然PM脂质或用PM脂质完全代替外脂质。对于每项新制剂，我们将将晶体与PM进行比较。有了最好的准备，我们将隔离绝对光谱和红外光谱。 新方向： 使用最原始的晶体，我们计划获得时间分辨的X射线衍射数据。使用导致我们获得分离的绝对红外光谱的相同过程，我们应该能够为光循环中每个中间体获得分离的衍射模式。如果成功的话，这将显示每个原子在蛋白质中的位置，因为它在整个膜上运输质子以形成电化学潜力。该信息应大大增加对质子泵（例如细胞色素氧化酶）的能量转导过程的理解。