STRUCTURE DETERMINATION OF ION CHANNEL PROTEINS

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There are about 80 different potassium channels, which are essential for living cells. These are classified as voltage-gated, inward rectifying, Calcium dependent, and prokaryotic. KcsA is a bacterial ligand gated potassium channel that opens upon stimulus by intracellular protons. KcsA has been successfully crystallized in various conditions for both the WT protein and for numerous mutants. Shaker channel, on the other hand, is a voltage gated K+ channel with similar architecture to KcsA, but Shaker presents a more difficult challenge for crystallization. Therefore, in one subproject, our aim is to study the structural and functional aspects of Shaker by constructing various KcsA-Shaker chimera proteins. Outer membrane protein F (OmpF) is the main gateway that allows antibiotic molecules to permeate Gram-negative bacterial cells. With the emergence of antibiotic resistance by virtue of decreased membrane permeability, it is necessary to find ways to increase antibiotic translocation across OmpF porin. Therefore, in a second subproject, we have crystallized OmpF in complex with various antibiotics. For the first time, we are able to experimentally visualize protein-drug interactions at the molecular level. Therefore, results of this work will give insights into the design of antibiotics with improved diffusional characteristics and target specificity. The ionotropic glutamate receptor ion channels (iGluRs) mediate excitatory responses at the vast majority of synapses in the brain and spinal cord. The binding of neurotransmitter molecules to the ligand-binding domain (LBD) of these receptors drives the opening of the transmembrane pore. These receptors assemble as tetramers. However, previous crystal structures have revealed only dimer complexes. Therefore, in this third project, we have solved the crystal structure of a novel GluR2 LBD dimer-of-dimers.