The long-term goal is to understand molecular basis for signal transduction at the structural level. Major focuses on three membrane proteins that form channels or pores across membranes, secondary focus is on a structural understanding of intracellular signalling by phosphorylation. With acetylcholine receptors, a ligand-gated ion conducting channel, the goal is a three-dimensional structure at a resolution sufficient for determining polypeptide folding and arrangement. Three-dimensional cubic crystals (space group P432, a = 257 A) for x-ray diffraction and electron microscopy, and two dimensional crystals for electron crystallography are to answer questions fundamental to understanding neurochemical mechanisms. Topography of chains is to be mapped by chemical labeling peptides already largely assigned by mass spectrometry, even of unpurified mixtures of cleavage fragments, plus or minus 0.2 Daltons up to 3,000 Daltons. Crystals of colicin Ia (C2221, a = 67, b = 178, c = 297 A) are to give a structure for a bacterial voltage gated channel-forming protein at 3.5 A resolution. Carbene labeling from photoactivable reagents and chemical labeling of accessible regions of either side are to delineate configuration within the membrane. Single channel conductance and mutagenesis are to uncover basic mechanisms controlling conductivity of this 70 kD ion channel. A 28 kD bacillus toxin is to give rise to a model for multimeric pore-forming complexes in membranes. This insecticidal hydrophobic protein crystallizes (P6122, a = 65, c = 165 A) and high resolution analysis will be completed using three solved derivatives, to 2.5 A resolution. A critical branch point switching enzyme in prokaryotes, isocitrate dehydrogenase, is for structure analysis in its P41212, a = 105.1, c = 150.3 to 2.5 A) active and phosphorylated inactive forms both crystallized. lt typifies ultrasensitive switching in biology. Structural analyses of high resolution site-directed mutant trypsin crystals permit the first true enzyme substrate complex, insights into structure and protein dynamics, and are to be used to understand mechanisms by which substrates can activate enzymes.
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