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. We have been able to obtain visible microscopic kinetic data with BR in its native membrane (PM) using a 50 micron squared sample size, which is the size of membrane crystals that we can grow. But, the number of photons passing through this tiny target is less than 1/31000th that which passes through a 1 cm diameter circle, which we used in previous non-microscopic studies. The only reason we can see kinetic changes is that both the CCD camera and image intensifier (ii) have enormous gain capabilities. But, the real signal emanates from relatively few photons. This results in high noise backgrounds and low signal to noise (SN) ratios. It is necessary to fit the raw kinetic data to 6 or 7 exponentials. With the 1 cm circle target, only a few thousand repeats were sufficient to attain a high enough SN for fitting of all of the kinetic constants with great accuracy. For the 50 micron sample, we calculate that up to 10 million repeats may be needed to fit the much noisier microscopic data. Based on some new developments, we may be able to improve this situation. The general procedure for growing BR membrane crystals yields crystals in the 50 micron size range. We have found that the BR in such a crystal displays a very similar kinetic profile to what we see with the BR in its native membrane. Phil Anfinrud of NIDDK is a collaborator who will perform the time-resolved X-ray diffraction kinetic studies on our crystals requires a larger size, which we have not been to able to grow. Recently, Jeorg LaBahn and colleagues at the Research Center Julich Institute of Complex Systems has published new approaches for growing the larger size crystals that we require. I have contacted Dr. LaBahn, and he would like to collaborate with us on the project. The larger crystals should also lead to our obtaining significantly higher SN ratios. If our target size is increased from 50 to 200 microns, there will be a 16- fold increase in the number of photons. To make this change we must design and construct new optical coupling between the microscope and ii. This work is in progress. B.) Studies on amyloidosis of amyloid beta (abeta) protein in Alzheimers disease (AD) The polymerization of monomers of amyloid beta through oligomers, fibrils, and plaques is recognized in the pathology of AD. Like many other polymerization phenomena, the process shows a lag phase followed by the formation of a nucleus which leads to logarithmic growth of the polymer which ends in a plateau. Originally it was thought that the pathology resulted from the large fibrils and plaques, but it is currently believed that the culprit is a small soluble oligomer. A likely candidate is the nucleus. It is also known that the predominant protein conformational change in the early steps is from random coil to -helix. The fibers and plaques are mainly beta sheet. Time-resolved IR measurements provide a means recording conformational changes at all stages of the polymerization. SVD (developed in my laboratory) should then allow us to obtain a time profile of these changes. Our goal is to identify the protein conformation and shape of the principal oligomer (i.e. nucleus) which begins the processes leading to the loss of axon function in AD. Our previous report showed that we can follow conformational changes that take place during abeta polymerization using infrared (IR) spectroscopy. It also described a team of researchers at NIH who will collaborate using atomic force microscopy (AFM), circular dichroism, dynamic light scattering, and electron microscopy. The plan is to perform IR experiments at NIST and all of the other studies at NIH. We plan to use the same abeta preparations, buffers, and incubation conditions at both sites. In order to align the kinetic time courses from both sites, we are building a duplicate set of laser static light scattering units that will be calibrated using the data collected by AFM. In this way, we should be able to synchronize IR time course data obtained at NIST with that of the AFM, circular dichroism, dynamic light scattering and electron microscopic studies at NIH . We have established that AFM is capable of following 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 was evident that fibrils were growing from the globular structures, which decreased in size and height. At 70 hr, we saw more complex entangled structures which could be plaque. The polymerization process involves hydrophobic attractions and hydrogen binding. It is favored by higher salt, lower pH, and higher temperature. Conversely, it is retarded by low salt, high pH, and low temperatures. By adjustment of these parameters, we can obtain prolonged lag phases, if needed, to help isolate the conformation at the beginning of the rapid phase of aggregation kinetics.

Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2013
Total Cost
$13,572
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Type
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