Development of unique instrumentation using novel approaches is, in many instances, necessary to the success of biomedical research. Areas of emphasis within our group are summarized below. 1) Energy-driven proton pumps are of major importance for energy transduction in living systems. The respiratory chain of animals as well, as single celled organisms, uses energy released from electron transport to oxygen to form an electrochemical gradient for protons, which is then used to synthesize ATP. In collaboration with Dr. R. Hendler, NHBLI, we have developed optical instrumentation that enables studies on the simple 26000 D, photon-driven, proton pump bacteriorhodopsin (BR) to pinpoint specific molecular transformations that generate the most voltage across the membrane. These studies support the view of the BR photocycle as consisting of two parallel cycles instead of a single photocycle that is favored by many researchers. Information obtained from this proton pump should help in understanding more complex systems such as mammalian cytochrome oxidase. Combined infra-red and optical spectroscopy has produced kinetic structural information that characterizes each step of the photocycle, and collaborative studies are underway with colleagues at NIST to produce single crystals of BR membrane protein. The ultimate goal of this project is to characterize the BR crystal using similar established approaches in concert with time resolved X-ray diffraction to obtain structural information at the atomic level, thus enabling an understanding of protein conformational changes that result in electrogenic transport of protons across the membrane. We have developed instrumentation to study the optical kinetics of the BR photocycle in approximately 1 microliter samples of membrane fragments. The BR is positioned between two 600 micron fused silica optical fibers, which carry the visible spectrum monitoring light produced by an Oriel xenon arc lamp, both orthogonal to a third fiber that carries the 532nm photolysing pulse generated by a Coherent 5W diode pumped second harmonic Nd:YAG laser that passes through a 100 micron hole in a spinning disk. The output monitoring fiber carries information on the spectral kinetic changes that occur following the photolysis pulse. These changes are identified by positioning the fiber at the entrance slit of an Acton .25m spectrograph, the output of which is monitored by either a Princeton Instruments CCD camera directly or indirectly after intensification by an Opelco-Videoscope image intensifier. This instrumentation acquires 524 successive spectra at 512 wavelengths in the visible spectrum covering the characteristic BR photocycle changes. The spectral changes in the photocycle are not uniform and comprise fast events on the sub-microsecond time scale to slower events with millisecond time scale changes. To accomplish this a staggered time schedule is used with time increments from 5 microseconds to 500 microseconds. Using the CCD directly presents a light limitation at the shorter exposure times and an image intensifier has been integrated into the system to overcome this limitation and to provide shorter sub-s exposure times. At present, in discussion with the manufacturer, we are trying to overcome an inherent internal protective mechanism that automatically decreases the image intensifier gain at high light levels to protect it from damage with the consequence that accurate data cannot be obtained. In a parallel effort with colleagues at NIST at the Center of Advance Research in Biotechnology, we are exploring the use of custom fiber optic cables, which we design and fabricate, that integrate into the visible/IR Bruker microscope and interface with an existing custom optical multichannel analyzer that we developed previously. This system will initially evaluate the feasibility of using this approach to study BR membrane crystals that are currently being grown by othher colleagues at CARB. We have already been able to collect spectra at an acceptable signal to noise ratio with spot sizes of 200 micron - a level that approaches the sub 100 micron size of the expected BR crystals. A manuscript that deals with the initial aspects of this work associated with interpretation of the visible/infra-red spectra in terms of structural changes and photocycle mechanism has been submitted to Journal of Physical Chemistry B. A complete description of the results is presented in Dr. Hendler's annual report. 2) Through a material transfer agreement with Sunstone Biophotonics, we are investigating the potential use of infra-red upconverting nanocrystals as labeling agents. In an initial pilot experiment, we have labeled sites on a glass slide that will capture analytes coupled to the upconverting nanocrystals. The perceived advantage of the upconverters over typical fluorescent labeling agents is that background fluorescence that limits the lowest level of detection should be virtually zero. In addition, many detectors have very low sensitivity in the infrared, used to excite the upconverters, thus reducing the need for stringent filtering requirements that are usually required to eliminate the excitation light. We are designing a microfluidic device based on PDMS technology that connects by a microfluidic channel a series of such sites each labeled with different capture ligands. If the PDMS inherent autofluorescence can be overcome using these upconverters, a versatile, easily fabricated, and easy-to-use separation device with high sensitivity that can be used in a research microscope should be achievable. 3) In collaboration with NINDS, a system has been designed for a wireless sensor to be placed on the wrist of Parkinson's disease patients to analyze limb motion. A prototype instrument is being evaluated. It will measure intensity, frequency and duration of tremors due to Parkinson's disease. The system will use a Shimmer mote platform consisting of a microcontroller, transceiver and sensors. 4) Together with colleagues from CIT, who designed and fabricated a visual stimulus, optical imaging, and control apparatus to study neural connectivity in fruit flies, we have collaborated with Dr. Chi-Hon Lee, NICHD, to integrate a near-ir laser into the optical path of the apparatus to provide a feedback stimulus to the fruit fly depending on its response to the visual stimuli. Near infrared is needed to prevent the feedback stimulus from being recognized as a visual stimulus by the fruit fly. 5) In a collaboration with Dr. B. White, NIMH, we developed a pilot instrument to simultaneously provide a thermal stimulus to a fruit fly using a focused 532nm laser and an infra-red camera to record the temperature time profile. The laser was focused in a region of thermally sensitive receptors that release hormones associated with wing unfolding. Although encouraging results were obtained, the results were inconclusive and a more rigorous approach is indicated for future experiments.
| 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 |
| Hendler, Richard W; Meuse, Curtis W; Braiman, Mark S et al. (2011) Infrared and visible absolute and difference spectra of bacteriorhodopsin photocycle intermediates. Appl Spectrosc 65:1029-45 |
| Kakareka, John W; Smith, Paul D; Pohida, Thomas J et al. (2008) Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer. J Biochem Biophys Methods 70:1116-23 |
