STRUCTURAL STUDIES OF A VOLTAGE-GATED SODIUM CHANNEL

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. The Spiller lab and our collaborators have been primarily engaged in completing the structure of a voltage-gated sodium channel. This project has suffered from poor crystal quality, and our efforts have focused on crystal improvement. Currently we are working with potential derivative datasets and a partial molecular replacement solution while we search for improved crystal forms. We have obtained crystals of NaChBac bound to an antibody Fab fragment and are currently working on phasing a 3.3[unreadable] dataset. This has proven to be quite a challenge due to the non-isomorphism and radiation sensitivity of the crystals. Phases derived from molecular replacement solutions of the Fab fragments are not sufficient to determine the structure of the channel, so we are working to improve the phases using both density modification and multi-crystal averaging as well as traditional heavy atom methods. We are also working to further stabilize NaChBac and improve the crystals by the semi-rational introduction of disulfides. Additionally, we have begun structural work working aimed at understanding cytoskeletal regulation. Two families of targets are being pursued: bacterial pathogens, and eukaryotic microtubule associated proteins. We have determined the structure of a putative bacterial microtubule modulating protein, VirA, although our subsequent work has cast doubt on the protein's function.