Mechanism of energy transduction by bacteriorhodopsin

细菌视紫红质的能量转换机制

• 批准号: 8746543
• 负责人: richard w hendler
• 金额: $ 1.36万
• 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/8746543
• 关键词: Alzheimer' s Disease; Amyloid beta-Protein; Amyloidosis; Animals; Atomic Force Microscopy; Axon; Bacteriorhodopsins; Behavior; Binding; Buffers; Caliber; Cell Nucleus; Circular Dichroism; Complex; Coupling; Data; Development; Electron Microscopy; Electron Transport; Electrons; Event; Fiber; Goals; Growth; Height; High temperature of physical object; Hydrogen; Image; In Situ; Institutes; Kinetics; Laboratories; Lasers; Lead; Measurement; Membrane; Membrane Proteins; Microscope; Microscopic; Molecular; Molecular Conformation; National Institute of Diabetes and Digestive and Kidney Diseases; Noise; Optics; Organism; Oxygen; Pathology; Phase; Photons; Polymers; Preparation; Procedures; Process; Protein Conformation; Proteins; Proton Pump; Protons; Publishing; Reporting; Research; Research Personnel; Respiratory Chain; Sample Size; Sampling; Shapes; Signal Transduction; Site; Sodium Chloride; Staging; Structure; System; Techniques; Test Result; Testing; Time; United States National Institutes of Health; Work; X ray diffraction analysis; X-Ray Diffraction; base; beta pleated sheet; charge coupled device camera; cold temperature; cytochrome c oxidase; design and construction; follow-up; improved; infrared spectroscopy; instrumentation; light scattering; monomer; novel strategies; polymerization; research study; voltage

Summary: A.) Development of instrumentation and procedures for comparing visible and IR kinetics of the BR photocycle in membrane protein crystals to that of in situ in tiny membrane fragments. We have been able to obtain visible microscopic kinetic data with BR in its native membrane (PM) using a 50 micron squared sample size, which is the size of membrane crystals that we can grow. But, the number of photons passing through this tiny target is less than 1/31000th that which passes through a 1 cm diameter circle, which we used in previous non-microscopic studies. The only reason we can see kinetic changes is that both the CCD camera and image intensifier (ii) have enormous gain capabilities. But, the real signal emanates from relatively few photons. This results in high noise backgrounds and low signal to noise (SN) ratios. It is necessary to fit the raw kinetic data to 6 or 7 exponentials. With the 1 cm circle target, only a few thousand repeats were sufficient to attain a high enough SN for fitting of all of the kinetic constants with great accuracy. For the 50 micron sample, we calculate that up to 10 million repeats may be needed to fit the much noisier microscopic data. Based on some new developments, we may be able to improve this situation. The general procedure for growing BR membrane crystals yields crystals in the 50 micron size range. We have found that the BR in such a crystal displays a very similar kinetic profile to what we see with the BR in its native membrane. Phil Anfinrud of NIDDK is a collaborator who will perform the time-resolved X-ray diffraction kinetic studies on our crystals requires a larger size, which we have not been to able to grow. Recently, Jeorg LaBahn and colleagues at the Research Center Julich Institute of Complex Systems has published new approaches for growing the larger size crystals that we require. I have contacted Dr. LaBahn, and he would like to collaborate with us on the project. The larger crystals should also lead to our obtaining significantly higher SN ratios. If our target size is increased from 50 to 200 microns, there will be a 16- fold increase in the number of photons. To make this change we must design and construct new optical coupling between the microscope and ii. This work is in progress. B.) Studies on amyloidosis of amyloid beta (abeta) protein in Alzheimers disease (AD) The polymerization of monomers of amyloid beta through oligomers, fibrils, and plaques is recognized in the pathology of AD. Like many other polymerization phenomena, the process shows a lag phase followed by the formation of a nucleus which leads to logarithmic growth of the polymer which ends in a plateau. Originally it was thought that the pathology resulted from the large fibrils and plaques, but it is currently believed that the culprit is a small soluble oligomer. A likely candidate is the nucleus. It is also known that the predominant protein conformational change in the early steps is from random coil to -helix. The fibers and plaques are mainly beta sheet. Time-resolved IR measurements provide a means recording conformational changes at all stages of the polymerization. SVD (developed in my laboratory) should then allow us to obtain a time profile of these changes. Our goal is to identify the protein conformation and shape of the principal oligomer (i.e. nucleus) which begins the processes leading to the loss of axon function in AD. Our previous report showed that we can follow conformational changes that take place during abeta polymerization using infrared (IR) spectroscopy. It also described a team of researchers at NIH who will collaborate using atomic force microscopy (AFM), circular dichroism, dynamic light scattering, and electron microscopy. The plan is to perform IR experiments at NIST and all of the other studies at NIH. We plan to use the same abeta preparations, buffers, and incubation conditions at both sites. In order to align the kinetic time courses from both sites, we are building a duplicate set of laser static light scattering units that will be calibrated using the data collected by AFM. In this way, we should be able to synchronize IR time course data obtained at NIST with that of the AFM, circular dichroism, dynamic light scattering and electron microscopic studies at NIH . We have established that AFM is capable of following the polymerization process. Under one particular set of conditions we compared images taken at 40 min, 2 hr, and 70 hr. The earliest images showed globular units of about 20 nm heights among much smaller units. At 2 hr, it was evident that fibrils were growing from the globular structures, which decreased in size and height. At 70 hr, we saw more complex entangled structures which could be plaque. The polymerization process involves hydrophobic attractions and hydrogen binding. It is favored by higher salt, lower pH, and higher temperature. Conversely, it is retarded by low salt, high pH, and low temperatures. By adjustment of these parameters, we can obtain prolonged lag phases, if needed, to help isolate the conformation at the beginning of the rapid phase of aggregation kinetics.

摘要：A。）开发仪器和程序，用于比较膜蛋白晶体中BR光循环的可见和IR动力学与微小膜片段中的原位。 我们已经能够使用50微米平方样品量在其天然膜（PM）中获得可见的微观动力学数据，这是我们可以生长的膜晶体的尺寸。但是，通过该微小靶标的光子的数量小于1/31000，该光子通过直径为1 cm的圆圈，我们在以前的非微观研究中使用了。我们能看到动力学变化的唯一原因是CCD摄像头和图像增强器（II）具有巨大的增益功能。但是，实际信号源自相对较少的光子。这会导致高噪声背景和低信号与噪声（SN）比率。有必要将原始动力学数据拟合到6或7个指数。有了1厘米的圆目标，只有几千重分足以达到足够高的SN，以符合所有动力学常数的精度。对于50微米样本，我们计算出可能需要多达1000万个重复序列来符合较嘈杂的显微镜数据。根据一些新的发展，我们可能能够改善这种情况。生长的BR膜晶体的一般程序在50微米尺寸范围内产生晶体。我们发现，这种晶体中的BR显示出与BR在天然膜中看到的非常相似的动力学曲线。 NIDDK的Phil Anfinrud是一名合作者，他将对我们的晶体进行时间分辨的X射线衍射动力学研究需要更大的尺寸，而我们尚无法成长。最近，研究中心朱利希复杂系统研究所的Jeorg Labahn及其同事发表了新的方法，以增强我们需要的更大尺寸的晶体。我已经联系了Labahn博士，他想与我们合作就该项目合作。较大的晶体也应导致我们获得明显更高的SN比。如果我们的目标尺寸从50微米增加到200微米，则光子数量将增加16倍。为了进行这种更改，我们必须在显微镜和II之间设计和构建新的光学耦合。这项工作正在进行中。 B.）关于阿尔茨海默氏病（AD）淀粉样β蛋白淀粉样蛋白（ABETA）的研究 AD的病理学认识到通过低聚物，原纤维和斑块通过低聚物，原纤维和斑块聚合。像许多其他聚合现象一样，该过程显示出滞后相，然后形成核，从而导致聚合物的对数生长，该聚合物在平稳期结束。最初认为该病理是由大的原纤维和斑块引起的，但目前认为罪魁祸首是一种小的可溶性低聚物。 可能的候选者是核。众所周知，早期步骤中主要的蛋白质构象变化是从随机线圈到-Helix。纤维和斑块主要是β表。时间分辨的IR测量值提供了在聚合的所有阶段记录构象变化的手段。然后，SVD（在我的实验室中开发）应允许我们获得这些更改的时间概况。我们的目标是确定主寡聚物（即细胞核）的蛋白质构象和形状，该过程开始了导致AD中轴突功能丧失的过程。 我们先前的报告表明，我们可以使用红外（IR）光谱法遵循Abeta聚合过程中发生的构象变化。它还描述了NIH的一组研究人员，他将使用原子力显微镜（AFM），圆形二色性，动态光散射和电子显微镜进行合作。该计划是在NIH和NIH的所有其他研究中进行IR实验。我们计划在两个地点使用相同的ABETA准备，缓冲和孵化条件。为了使两个站点的动力学时间课程保持一致，我们正在建立一套重复的激光静态光散射单元，该单元将使用AFM收集的数据进行校准。这样，我们应该能够将NIST获得的IR时间过程与AFM，圆形二色性，动态光散射和NIH的电子显微镜研究同步。 我们已经确定AFM能够遵循聚合过程。在一组特定条件下，我们比较了40分钟，2小时和70小时拍摄的图像。最早的图像显示在较小的单位中的球状单元约为20 nm。在2小时时，很明显，纤维从球状结构中生长，尺寸和高度降低。在70小时时，我们看到了可能是斑块的更复杂的纠缠结构。聚合过程涉及疏水景点和氢结合。它受到较高盐，较低的pH和较高温度的青睐。相反，它被低盐，高pH值和低温所阻碍。通过调整这些参数，我们可以在需要时获得延长的滞后相，以帮助隔离聚集动力学快速阶段开始时的构象。