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. We are continuing to study the detailed mechanism of ion transport in a CLC-type Chloride transport protein. This work involves the examination of crystal structures of functionally informative mutant proteins. The two basic questions we are now engaged in answering - and that will be occupying us for the next cycle, I predict - concern (1) the movement of protons through this transport protein and (2) the nature of the Cl-/H+ exchange pathways. The protein, CLC-ec1, is a Cl-/H+ exchanger from E coli. It catalyzes electrogenic, stoichiometric trasnmembrane exchange of Cl- for H+ via an unknown transport mechanism. The protein shows binding sites of 3 Cl ions lying in a roughly transmembrane orientation, so we have a pretty good idea of the anion pathway. But how movement of Cl- ions along this pathway is coupled to H+ transport in the opposite direction is unknown. As a result of structure-and-function work of the past year, some of it at NSLS, we have identified two key glutamate resdiues required for tranferring protons from the aqueous solutions into the protein interior - one facing the extracellular solution and one the intracellular. These two residues are separated by about 20 angstroms, and we are now seeking protonatable sites between these that protons """"""""hop"""""""" along as they move across the membrane through this protein. In order to make mechanistic sense in this study, we require three basic types of information: (1) electrophysiological behavior of the mutants, (2) crystal structures of the mutants, and (3) positions and occupancies of the halide-binding sites, via anomalous diffraction arising from Br- ions substituted for Cl-. The prtooein crystalizes in combination with a Fab fragement and diffracts to 3.0-3.5 A resolution. We are endeavoring to improve this, but it is adequate for attacking the questions we are currently addressing.
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