9603935 Cross The knowledge gained from the gramicidin studies will now be used to face the challenges associated with a biosynthetically prepared protein, M2 protein, studied as a dilute species in a lipid bilayer. Improvements in sensitivity will come from modications in the sample geometry, increased field strength and improvements in the experimental protocols. Linewidths in the M2 samples are primarily dominated by sample heterogeneity which through adjustments in sample preparation techniques can be addressed and there is also reasonable expectations that linewidths (in ppm) at least in some cases will decrease the increasing field strength. The mathematical challenges will involve the characterization of imperfect alpha-helices that are prevalent in membrane proteins. The work will take this solid state NMR method that utilizes orientational rather than distance constraints and apply it to a "real" protein system. High technology methods involving Nuclear Magnetic resonance (NMR) spectroscopy will be developed to solve the three-dimensional structures of macromolecules. Typically such structures are determined by diffraction methods applied to crystallized samples, but there are many important macromolecules that are not crystallizable. This unique NMR approach uses samples that are uniformly aligned, in other words they are oriented in one dimension, while crystals involve three-dimensional order. The molecular system that will be used for demonstrating these methods is protein that forms an ion channel in membranes. The NMR data obtained will be analyzed by new mathematical approaches that will be generally applicable to many molecular systems. Overall, the development of this structure determination approach will open new avenues of research for a great many types of molecules that are not easily crystallized. The work will provide great training opportunities for graduate students and postdocs in an interdisciplinary environment where molecular biology, synthetic chemistr y, NMR spectroscopy, NMR hardware development, molecular modeling and mathematical analysis occur in the confines of this research effort.
