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 tiny purple membrane fragments (PM). The assumption in using X-ray crystal protein structures for elucidating in vivo functionality is that the protein conformations are the same in both environments. But, satisfactory crystals can only be grown below pH 5.9, whereas in nearly all PM studies, a pH 7 near has been used. All published X-ray structures used these low pH crystals. In our 2014 annual report, we described a procedure where we slowly moved crystals from pH 5.9 to 7.0 in steps of 0.2 pH units. In order to compare the protein conformations in pH 5.9 crystals with that in membranes at pH 7, we used the Amide I and Amide II frequency regions in infrared spectroscopy (IR.). In this way, we found that the pH 5.9 crystal BR is not a valid conformational model for the pH 7 membrane BR. Specifically, both Amide IR profiles were significantly broadened in the crystals. Earlier findings from our laboratory in collaboration with Ira Levin of NIDDK showed that such broadening was the result of greater mobility of peptide bonds leading to interactions with neighboring electromagnetic fields. We sent both our pH 5.9 and pH 7 crystals to Brookhaven National Laboratories, to obtain structures and atomic resolutions. Our 6-week old pH 5.9 crystals had 2.7 Angstrom resolution compared to 6.5 Angstrom for the pH 7 crystals. Fresh pH 5.9 crystals have 1.3 Angstrom resolution. These findings make it imperative that crystal and membrane functionalities both be compared at pH 5.9. This presents a problem. In order to use my linear algebraic mathematical procedures to obtain pure isolated structures for each photocycle intermediate, the precise kinetic model must be known. We have published precise (different) modes that operate at pH 5, 7, and 9. The pH 7 model contains 2 linear chains and is fit by 7 exponentials. The pH 5 model contains 3 linear chains and is fit by 9 exponentials. We have obtained evidence that strongly indicates that kinetic models at pH 5.9 and pH 7 are very close, if not identical. We must now accumulate a large number of repeat experiments to increase the signal to noise ratio to allow accurate fitting of the 7 kinetic constants. Once this is done, it is time to obtain time-resolved Laue X-ray kinetic data that will allow the isolation of each separate atomic structure and see how protein conformational changes work to move protons across the membrane to form an electrochemical potential. Our X-ray crystallographer Joerg Labahn is planning to obtain the Laue data. B.) Studies on amyloidosis of amyloid beta (abeta) protein in Alzheimers disease (AD). As described in our previous report, our focus is on identifying the small soluble oligomer that is currently thought to initiate deterioration of brain function in AD. In the 2014 report we presented reasons for our belief that the pathogenic oligomer may be a small α-helical peptide that can open hydrophilic channels that dissipate membrane potential. We suspect that this structure appears at the end of the initial lag phase in polymerization, which is also the beginning of the logarithmic phase. In order to pinpoint the various stages of polymer growth, one must know the kinetic mechanism of the process. As stated in Goals and Objectives, the reason we became involved with abeta polymerization is because of mathematical procedures developed in my laboratory (SVD) that can identify steps involved in sequential linear reaction paths. We present our first findings here. Using AFM time-resolved images, we identified several stages that suggest the following likely 4 step scheme ........k1.............k2..............k3..............k4... G -------->F1--------->F2--------->F3----------->F4, where G represents a globular structure, and the other 4 states as increasingly bigger, unique structures. Kinetic constants for each step are shown. Expressing the scheme in a set of ordinary differential equations, allowed us to obtain the time curves for growth and dissipation of each component from starting G to final F4. In addition to the time profiles, SVD requires spectra that represent each component. We used 5 quantifiable parameters in such 'spectra' (viz.) 1. Height of G; 2. Width of G; 3. Maximum length; 4. Width of fiber; 5. Maximum width. From this, we produced a test matrix set (A) of raw data. SVD obtained spectral eigenvectors (U), singular values (S), and kinetic eigenvectors (V). From V, we determined the number of steps and retrieved the known correct kinetic constants which showed the times of the transitions and rates of growth of the individual structural intermediates, as well as the clear transition of lag to logarithmic growth. Our next step is to apply this approach to actual raw data from atomic force microscopy where the number of steps and kinetic constants are unknown. We will then use infrared spectroscopy and circular dichroism to determine protein conformation at each step, and Thioflavin T fluorescence to verify the exact point of transition from lag to log phases.
|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|