The objective of this proposal is to understand the function of the mammalian water channel CHIP28 in the bilayer from its high-resolution 3- dimensional (3-D) structure and structural modulations determined by electron crystallography. CHIP28 which facilitates osmotic water transport across erythrocyte and various water-permeable epithelial cell membranes functions as a channel selective to water excluding small ions and solutes.
Our specific aims are listed below.
AIM I. Determine the projected structure of the CHIP28 molecule in the bilayer. We will extend the knowledge of the projection structure of CHIP28 from our current low-resolution (14 Angstroms) map by computing maps to progressively higher resolution (8 Angstroms and better) based on analysis of images and diffraction patterns collected from unstained frozen-hydrated specimens. These maps will provide finer definitions of the molecular envelope, intermolecular packing and constraints on the path of the polypeptide chain and possible description of elements of secondary structure.
AIM II. Determine the 3-D structure of the polypeptide chain of CHIP28 from reconstructions based on data from tilted specimens. 3-D density maps will be calculated based on diffraction patterns and images collected from frozen-hydrated specimens tilted in the microscope. These maps generated at progressively higher resolution will elaborate in 3 dimensions the secondary structure and tertiary folding of the polypeptide chain as it traverses the bilayer. The ultimate goal is the postulation of a chemical model for the regulation of water transport from the folding and packing of the polypeptide chain.
AIM III. Examine the structural basis for the inhibition of the water- transporting property of CHIP28 by mercurial compounds. As a first step towards understanding function of CHIP28 from its modulations, the structural role of the mercurial pharmacological inhibitor will be studied. Electron diffraction patterns, initially, in projection will be used at the highest possible resolution to quantitate structure changes upon mercurial binding to the known single extracellular CYS189 residue. CHIP28 belongs to the so-called MIP super-family of channel proteins from diverse organisms which likely share similar structure/function correlates. Thus the proposed research will add to elucidation of basic mechanisms of transmembrane signaling. The selective expression of CHIP28 and homologous water channels in epithelial cells believed to be involved in fluid absorption and/or secretion makes it an extremely important pharmacological target. Thus high-resolution 3-D structure will potentially have impact on structure-based drug design.
| Ren, Gang; Quispe, Joel; Leppla, Stephen H et al. (2004) Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study. Structure 12:2059-66 |
