STRUCTURE DETERMINATION OF A PROKARYOTIC CYCLIC NUCLEOTIDE-MODULATED ION CHANNEL

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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. To date there are few membrane proteins with available high-resolution 3D structure. We have identified and functionally characterized a prokaryotic homolog of eukaryotic cyclic nucleotide-gated channels (Nimigean et al., 2004, JGP), for which family there is no atomic resolution structure available. The eukaryotic CNG and HCN channels are crucial players in signal transduction. The CNG channels play important roles in visual and olfactory signal transduction while the HCN are the pacemaker channels in the heart and brain. A high-resolution structure of this ion channel type will be a significant advance in the field. We have been working for the past year on further characterizing the prokaryotic channel homolog in addition to trying to obtain diffraction quality crystals of the native protein and various constructs/mutants. We have obtained crystals that we tested previously both at home sources and at synchrotron beams. The diffraction quality was extremely poor at home source (7-20 A resolution) and improved significantly at synchrotron beams (3.5-8 A). Unfortunately, so far the quality of the data precluded us from solving the atomic structure. We think we have now optimized these crystals by using a slightly altered construct.