The long-term goal of the laboratory is to gain a structural understanding of the molecular organization and function of biological macromolecules at the biological membrane interface. Our focus of this study is to understand physical mechanisms of ion selectivity, conductance regulation, channel gating of ion channel proteins, and the structural basis of molecular specificity for oligomerization by X-ray crystallography. The first specific goal is to determine the three-dimensional structure of the tetramerization domain of Shaker potassium channel. We have obtained three-dimensional crystals of the soluble domain of this channel that diffract X-ray beyond 2.0 Angstroms. Crystals belong to the space group I4 with one subunit molecule per asymmetric unit. We have also obtained three-dimensional crystals of the soluble, domain of a channel from a different subfamily of potassium channel Shaw that diffract X-ray to about 3.0 Angstroms. The second specific goal is to determine the atomic structure of an inwardly rectifying potassium channel ROMK1 from rat kidney. Towards this goal, the underlying hypothesis of our novel strategy is that the lipid- facing exterior of a channel protein can be mutated systematically without perturbing its channel function. Site-directed mutagenesis was used as a perturbation method in order to identify the lipid-facing residues. Based on this study, simultaneous changes have been introduced at sites in its transmembrane lipid-facing exterior of the channel, which do not alter the channel function. The surface of this exterior-modified channel has a sequence motif known to form a specific helix-to-helix interaction, through which the exterior-modified channel itself is solubilized by binding of synthetic amphipathic peptides to the channel exterior in the aqueous phase without the use of detergents. Understanding the structural basis for tetramerization will provide an essential knowledge on the physical nature of channel diversity. From the atomic model of the ROMK1 channel, we seek to establish the exterior- modification strategy that will be generally applicable to structural studies of a variety of integral membrane proteins such as ion channels and transmembrane receptors.
Showing the most recent 10 out of 11 publications
