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.

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Hendler, Richard W; Meuse, Curtis W; Gallagher, Travis et al. (2015) Stray light correction in the optical spectroscopy of crystals. Appl Spectrosc 69:1106-11
Hendler, Richard W; Meuse, Curtis W; Smith, Paul D et al. (2013) Further studies with isolated absolute infrared spectra of bacteriorhodopsin photocycle intermediates: conformational changes and possible role of a new proton-binding center. Appl Spectrosc 67:73-85