Mechanism of energy transduction by bacteriorhodopsin

细菌视紫红质的能量转换机制

基本信息

批准号：
7734943
负责人：
richard w hendler
金额：
$ 0.42万
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

来源：
https://reporter.nih.gov/project-details/7734943
关键词：
Amides Animals Area Bacteriorhodopsins Behavior Binding Biological Assay Biomedical Engineering Buffers Cells Characteristics Charge Collaborations Collection Complex Condition Crystallization Data Desiccation Devices Dryness Electron Transport Equilibrium Equipment Event Extinction (Psychology)Goals Hour Image In Situ Institutes Joints Kinetics Laboratories Lasers Learning Length Light Link Measures Membrane Membrane Proteins Meta-Analysis Microscope Molecular Monitor Movement Noise Numbers Optics Organism Oxygen Photons Problem Solving Procedures Process Proteins Proton Pump Protons Quality Control Rate Research Personnel Respiratory Chain Running Sampling Schedule Signal Transduction Sodium Chloride Solutions Spectrometry Spectrum Analysis Staging Standards of Weights and Measures Structure System Technology Time United States National Institutes of Health Water Work X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction aqueous bioimaging charge coupled device camera cytochrome c oxidase desire detector evaporation hypodermic needle inorganic phosphate instrument pressure protein function time interval vapor voltage

项目摘要

Our efforts the past year have been to define experimental conditions to allow simultaneous, parallel kinetic studies on the same sample of BR using both IR and optical spectroscopies. There have been many challenges. 1.) Water is an essential component for normal photocycle behavior, but because of its intense IR absorbance in the same region as the amide components of proteins, only about 0.2 microliters, or less can be present in the <0.2 mg BR sample. The collection time for IR data takes several hours during which time thousands of laser flashes are used. With this small amount of H2O, evaporation could result in desiccation and perturbations in kinetic behavior. We have found a solution to this problem. A special IR cell is used that has a small ridge drilled along its outer edge and a tight-fitting cap. An aqueous solution is placed in the outer ridge to serve as a reservoir to maintain an adequate steady-state vapor pressure in a 120 micron space above the sample in the center of the cell. The extinction coefficient for the characteristic absorbance (A) of H2O is used to quantify the actual H2O content of the sample. This amount of H2O results from the equilibrium between the vapor pressure over the reservoir and the sample. By trial and error we have determined a concentration of phosphate solution in the reservoir that provides an optimal amount of H2O in the sample. Even at this concentration of H2O in the sample, the remaining (A) due to H2O decreases the transmittance of IR light to a point that results in significant background noise. We have determined that a sufficiently high signal to noise ratio to allow the required fitting of 6 exponential constants to the data requires the pooling of several hundred samples. 2.) Because the kinetics of the BR photocycle are markedly influenced by pH, it is essential to know the pH of the sample being assayed. But, the sample volume is only about 0.2 microliters, spread over an area of about 1 cm2 and no pH probe can be used for such a sample. Furthermore, the pH of the initially dilute sample can not be assumed to hold upon the evaporation of most of the H2O. This pH problem can not be solved by increasing buffer strength, because the dried salt residue will retain a higher H2O content upon equilibration with the reservoir. We have solved this problem by using a micro-combination probe contained in a hypodermic needle than can measure pH in a volume of near 1 microliter. We follow the change in pH as a sample of 50 microliters is taken to dryness and have learned what pH must be established in the beginning in order to achieve the desired pH at near dryness. To guarantee that the IR sample has not deteriorated during its long run because of desiccation or laser damage, we use the optical spectrometer on the identical sample both at the beginning and end of each 100-repeat run used for pooling the data. The optical kinetics provides a much higher signal to noise ratio, uses only a single laser flash for each entire photocycle and takes only about 10 minutes for 400 repeats. The next stage of the project is to obtain a super matrix of spectral data with columns representing absorbances at 600 to 900 discrete time points and rows representing absorbances at about 90 discrete optical wavelengths in the top part and at about 600 to 1000 discrete IR wave numbers at the bottom. We will then apply the mathematical, analytical procedures developed in our laboratory to try to obtain, for the first time, isolated absolute IR spectra for each intermediate. These data should provide important structural and proton-binding information to better understand conformational changed linked to electrogenic proton-pumping. New directions: We have initiated a new project in collaboration with the National Institute of Biomedical Imaging and Bioengineering (NIBIB) and the National Institute of Standards and Technology (NIST) to build an instrument that can obtain entire optical and infrared spectra (IR) very rapidly (every few microsec) from a single crystal of membrane protein. We will start with the energy-transducing proton pump, BR. No instrument capable of performing such studies has been previously described. There are many reasons to have such an instrument. 1.) Quality control to verify whether a membrane protein functions the same in the crystal as in situ in the membrane. 2.) As a guide to alter the crystallization procedure to produce crystals more closely related to in situ function. 3.) To produce valid crystals to be used in time-resolved X-ray crystallography in order to obtain isolated atomic structures for each intermediate in the proton-pumping procedure so that the overall process can be better understood. The optical spectrometry approach will be developed at NIH and the IR spectroscopy at NIST. When each part is successfully working the optical equipment will be moved to NIST to integrate the joint system. Eventually, both NIH and NIST can have the same combined working system. NIST has already ordered a new IR spectrometer and a special microscope to focus both visible and IR light on the crystal sample. During the year, we have determined that the optical system instrument will be built around a charge-coupled device (CCD) camera with an attached spectrograph. The CCD has a photon detector that contains 1024 rows of 512 pixels each. Each row can record a spectrum of 512 wavelengths and each spectrum can be obtained at a different point in time. NIBIB purchased this CCD/spectrograph unit from Princeton Instruments with the understanding that it could perform the rapid spectral acquisitions required. We have since learned that no one has actually used the CCD in the way we intend, and the instrument as delivered can not. The problem is that in a kinetic cycle consisting of several sequential intermediates with different time constants, it is necessary to use a staggered collection schedule which starts with many closely spaced time points (every few microsec) and then continuously lower the collection rate ( i.e. to 500 microsec) towards the end. The device can alter the collection schedule as desired, but the line of pixels remains exposed to the monitoring light for the entire length of each time sequence. Instead of obtaining spectra at sharp points in time, there is an accumulation of photons over the entire period of exposure (i.e. 500 microsec for the slower collection times). We have found a way to remedy this problem. An image intensifier can be placed between the exit of the spectrograph and entrance to the CCD camera. A sharp square wave gate can be used to make the intensifier serve as a programmable shutter that will admit light only for an instant at the end of each timing interval. We plan to obtain this intensifier and integrate it into the system.

过去一年，我们的努力是定义实验条件，以便使用IR和光谱镜同时在同一BR样品上同时进行平行动力学研究。有很多挑战。 1.）水是正常光循环行为的重要组成部分，但由于其与蛋白质的酰胺成分在同一区域中具有强烈的IR吸光度，因此只有大约0.2微透明剂或更少的含量在<0.2 mg br样品中存在。 IR数据的收集时间需要几个小时，在此期间，使用了数千个激光闪光。使用少量的H2O，蒸发可能导致动力学行为的干燥和扰动。我们找到了解决这个问题的解决方案。使用了一个特殊的IR电池，其沿其外边缘钻了一个小山脊和一个紧身的帽子。将水溶液放在外脊中，作为储层，以在细胞中心的样品上方的120微米空间中维持足够的稳态蒸气压。 H2O的特征吸光度（a）的灭绝系数用于量化样品的实际H2O含量。 H2O量是由于储层上的蒸气压与样品之间的平衡而产生的。通过反复试验，我们确定了储层中磷酸盐溶液的浓度，该溶液在样品中提供了最佳量的H2O。即使在样品中的H2O浓度下，由于H2O引起的其余（a）也会将IR光的透射率降低到导致明显的背景噪声的点。我们已经确定一个足够高的信号与噪声比，以允许将6个指数常数与数据拟合，需要汇总数百个样本。 2.）由于BR光循环的动力学受到pH的显着影响，因此必须知道要测定的样品的pH值。但是，样品体积仅为0.2微升，分布在约1 cm2的面积上，并且该样品无需使用pH探针。此外，不能假定最初稀释样品的pH值保持在大多数H2O的蒸发上。无法通过增加缓冲液强度来解决此pH问题，因为干燥的盐残基将在与储层平衡时保留更高的H2O含量。我们通过使用皮下注射针中包含的微型组合探针解决了这个问题，而不是可以在接近1微晶的体积中测量pH。我们遵循pH的变化，因为将50微升的样本归为干燥，并了解了一开始必须建立哪些pH值，以便在几乎干燥时实现所需的pH。为了确保IR样品在长期内由于干燥或激光损伤而没有恶化，我们在每次用于汇总数据的100个重复运行的开头和结尾都使用相同样品的光谱仪。光学动力学提供了更高的信号与噪声比，每个光循环仅使用一个激光闪光，仅需10分钟即可完成400个重复。该项目的下一个阶段是获得光谱数据的超级矩阵，其列代表600至900个离散时间点的吸光度，而行则表示在顶部的90个离散光波长，在顶部约为90个离散光波长，底部约为600至1000离散的IR波数。然后，我们将应用实验室中开发的数学，分析程序，以尝试首次获得每个中间体的绝对IR光谱。这些数据应提供重要的结构和质子结合信息，以更好地理解与电源质子泵送相关的构象变化。新方向：我们已经与国家生物医学成像和生物工程研究所（NIBIB）和国家标准技术研究所（NIST）合作启动了一个新项目，以从膜蛋白的单晶蛋白质中迅速获取一个可以非常快速地（每几个microsec）获得整个光学和红外光谱（IR）的乐器。我们将从能量传递的质子泵开始。先前尚未描述任何能够进行此类研究的仪器。拥有这样的乐器有很多理由。 1.）质量控制以验证膜蛋白在晶体中的作用是否与膜原位相同。 2.）作为改变结晶过程的指南，以产生与原位功能更紧密相关的晶体。 3.）产生有效的晶体，用于在时间分辨的X射线晶体学中使用，以便为质子泵送过程中的每个中间体获得分离的原子结构，以便更好地理解整个过程。光谱法将在NIH和NIST处开发IR光谱法。当每个部分成功使用时，光学设备将被移至NIST以整合关节系统。最终，NIH和NIST都可以具有相同的合并工作系统。 NIST已经订购了一个新的红外光谱仪和一个特殊的显微镜，以将可见光聚焦在晶体样品上。在这一年中，我们确定光学系统仪器将围绕带有附着光谱仪的电荷耦合设备（CCD）摄像头构建。 CCD具有一个光子检测器，每个检测器包含1024行，每行512像素。每行都可以记录512波长的频谱，并且可以在不同的时间点获得每个光谱。尼比布（Nibib）从普林斯顿仪器（Princeton Instruments）购买了此CCD/光谱仪单元，并了解它可以执行所需的快速频谱采集。从那以后，我们了解到，没有人实际上以我们打算的方式使用了CCD，而交付的仪器则不能。问题在于，在动力学循环中，由几个具有不同时间常数的顺序中间体组成，有必要使用交错的收集时间表，该计划以许多紧密间隔的时间点（每几个microsec）开始，然后不断降低收集率（即500 microsec）。该设备可以根据需要更改收集时间表，但是在每个时间顺序的整个长度上，像素线仍然暴露于监视灯。在整个暴露期间，没有在尖锐的时间点获得光谱的光子（即较慢的收集时间的500 microsec）。我们找到了一种解决这个问题的方法。可以将图像增强器放置在光谱仪的出口和CCD相机入口之间。锋利的方波门可用于使强化器用作可编程快门，该快门只能在每个正时间隔结束时立即接收光。我们计划获取此增强器并将其集成到系统中。