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. The assumption in using X-ray crystal protein structures for elucidating in vivo function is that the protein conformations are the same in both environments. The findings we report here with BR show this assumption is not always valid. The pH optimum for obtaining crystals is often quite different than that for normal in vivo functionality. BR crystals are grown near pH 5.9, but in vivo, a pH near 7 is more realistic. The only published X-ray structures have been obtained for crystals grown below pH 6. Crystals cannot be grown at pH 7. We developed a method to slowly move crystals from pH 5.9 to 7.0 by acclimatization in steps of 0.2 pH units up to pH 7. In order to compare the protein conformations in pH 5.9 crystals with that in membranes at pH 7, we used infrared (IR.) To do this we had to solve another problem. Crystals must be hydrated to maintain their integrity. But, free water strongly absorbs IR radiation. If samples at pH 5.9 and 7.0 have different free water contents, subtle changes in conformation will be overshadowed by the water difference spectra. We developed a mathematical procedure to subtract amounts of excess water spectrum to produce minimal constant levels for all samples. In this way, we found that the pH 5.9 crystal is not a valid model for the pH 7 membrane. On the basis of these findings we have sent our pH 7 crystals to the cyclotron at Brookhaven National Laboratories, to obtain the first structures for crystals at pH 7. In the course of both IR and visible spectroscopies, we recognized another problem that has not been properly dealt with. The BR crystal is hexagonal. To use the whole crystal, some light reaches the detector without passing through the crystal. This phenomenon (stray light) produces distorted and unreliable spectra. We have developed a new procedure that allows the use of the entire crystal and corrects the distortions caused by stray light. A paper describing this new procedure has been submitted for publication to Applied Spectroscopy. Another problem mentioned in the previous report was that with the 50 micron crystals, usually obtained, it was not possible to obtain sufficient rapid time-resolved Laue X-ray diffraction data. The small crystals also limited the amount of rapid visible data for fitting all 7 of the kinetic constants in the photocycle. These are needed to obtain the separate X-ray structures for each photocycle intermediate. Two German crystallographers have recently described procedures that produce crystals up to 200 microns in size. Fortunately, both of them have agreed to collaborate with us and supply larger crystals as needed. We have already modified the visible optics of our microscope to work with the larger crystals. B.) Studies on amyloidosis of amyloid beta (abeta) protein in Alzheimers disease (AD) The abeta monomer is very similar in size and amphiphillic nature to a group of antibacterial peptides known as magainins. My laboratory has shown that magainins can polymerize into α-helical configuration that can penetrate cell membranes forming aqueous channels that dissipate membrane potential. Current thinking in AD is that a small soluble oligomer is the pathogen that destroys brain neural function. Our working hypothesis and focus of approach is that at the end of the initial lag phase in polymerization, the pathogenic oligomer with α-helical configuration is formed. At the same time, this oligomer promotes rapid growth leading to fibrils and plaques. There are publications indicating a transient α-helical phase at the end of the lag phase. We have developed a new approach for following time-dependent changes in secondary structure based on singular value decomposition of circular dichroic data. Our plan is to follow polymerization using simultaneous measurements of AFM (atomic force microscopy) infrared and circular dichroic spectroscopies, as well as dynamic light scattering to provide more solid evidence implicating the α-helical oligomer as the initial pathogen and nucleus for formation of plaques. Our previous report showed that AFM is capable of imaging 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 appeared that fibrils were growing from the globular structures, which decreased in size and height. At 70 hr., we saw more complex entangled structures which resemble plaque. Traditionally, AFM is performed with a dried sample on a mica support. The dried sample contains buffer salts which limits resolution of abeta monomers and small oligomers. We have been exploring a new form of AFM that uses a wet sample. Not only does this eliminate the dried salt problem, but it allows following of the whole live polymerization process in real time. Our results obtained with wet AFM, presented in the August 7 NIH Summer Research Program Poster Day, were sharper than any we have seen in published reports. Preliminary findings indicate that a trimer, rather than a monomer binds to the growing fiber. Furthermore, in the beginning (lag phase), there is very slow growth of the fibers. Then, a supporting linear structure appears, to which fibers attach. Polymerization then speeds up and new 'trimers'attach to the distal untethered end of the growing chain. We plan to proceed using both the wet and dry AFM procedures, as well as IR and circular dichroism to follow changes in secondary structure and the possible role of a soluble α-helical oligomer in AD.
|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|