Support is requested for a collaborative study to provide structure function information for the CHIP28 water channel. The approach is to use steady-state and time-resolved analysis of intrinsic tryptophan fluorescence of a single tryptophan, single cysteine-CHIP28 mutant to measure selected site-site distances and nanosecond segmental motion in functional water channels reconstituted into proteoliposomes. The mutant proteins will be expressed in baculovirus-infected Sf9 cells, purified and reconstituted into liposomes. The single reactive sulfhydryl will be labeled with coumarin or bimane chromophore.
The specific aims are: 1) to analyze the membrane structure of Chip28 and the packing arrangement of helical domains; and 2) to measure the nanosecond segmental motions of CHIP28 to test if the mercurial inhibition of water channels is associated with a change in CHIP28 dynamics. Rotational parameters will be compared to water transport function for each mutant, and effects of temperature and mercurial inhibition will be examined.
