Using our own linear algebraic analytical methodology we were successful, for the first time, in obtaining clean isolated absolute optical and IR spectra for each intermediate of the bacteriorhodopsin (BR) photocycle. With these, we could obtain, for the first time, difference spectra for each transition between consecutive intermediates. All previous difference spectra were for the intermediate minus the ground state, and were obtained with non-pure intermediates. Our earlier studies demonstrated that instead of a single homogeneous photocycle containing two forms of the M-intermediate in sequence, there is a heterogeneous set of parallel cycles with one form of M in each. We label the cycles fast and slow based on whether they contain the faster or slower turning-over form of the M-intermediate. Mark Braiman, an expert in the interpretation of BR IR spectra, accepted our invitation to help interpret the wealth of new information we have obtained, and most of the past year has been devoted to this effort. I will briefly list two of the most important new findings. 1. The fast and slow cycles serve different functions, the former primarily converts photo energy into a delta pH, while the latter forms a membrane potential (delta psi). 2. A long-standing quest has been to identify the source of the proton release group (PRG) that discharges the pumped proton to the external medium. One predominant view is that the PRG is a crystalline protonated water cluster. Our findings do not support this idea. On the other hand, the kinetics of deprotonation of ARG82 are entirely consistent with it being the PRG. Furthermore, ARG82 deprotonation occurs only in the M-fast cycle. The formation of delta psi is based on the longer retention of the proton on the external surface before its loss to the medium. In addition to these, many other interpretations, based on the cleanly isolated intermediates of the two cycles were made. This work has been written up and submitted to the J. Phys. Chem, where it is under review. B.) Development of instrumentation for studying optical kinetics of the BR photocycle in single membrane/protein crystals and tiny membrane fragments. The instrumentation is built around a Princeton Instruments charge-coupled device (CCD) camera and integrated spectrograph. With this device, we can acquire 524 successive spectra at 512 wavelengths on a time scale from microsec to ms. To cover a range of 60 ms in 524 time points requires using a staggered time schedule with increments from 0.005 up to 0.5 ms. This presents a problem because the number of photons acquired by the detector is proportional to the exposure time. We planned to obtain even exposure times by using a gated image intensifier (ii) with pulses at or below 0.001 ms. However, after much time at trying to make this work, we learned that no one has ever done this before. The problem is that to protect the sensitive photo-detector from damage an excessive photon flux, an internal circuit automatically decreases the ii gain. With time intervals of 0.01 ms, we can not obtain accurate data. In discussions with the manufacturer there may be a fix, which we are exploring. In the meantime, we are in the process of designing ways to proceed with the CCD device alone if we must. 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. We have acquired a new Bruker IR spectrometer and microscope. This should allow us to work with either single membrane protein crystals or tiny fragments of BR in situ in its native membrane. Initially neither we nor the experts from Bruker could make the system perform as it should. It seems that we are the first ones to work with this new equipment. Recently, with the help of Bruker specialists, we have been able to obtain data with a target size of 100 microns. We are now trying to decrease this target size to 50 microns, But, alignment requirements become very demanding for this. Our crystallographer collaborator at NIST has been successful at growing BR membrane crystals. Procedures for obtaining membrane protein crystals most often use exposure to detergent, which our previous work has shown will alter the normal kinetics of turnover. Although not used, procedures for obtaining crystals in the absence of detergent have been described. Our crystallographer has started to repeat this approach, and very small crystals are beginning to grow.

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National Heart, Lung, and Blood Institute
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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