Professor Eduardo Perozo
Department of Molecular Physiology and Biological Physics and Center for Structural Biology
University of Virginia Health Sciences Center
Charlottesville, VA

Monday, May 2, 2005
3:00 pm (CT)
3269 Beckman Institute

Abstract

The fundamental processes that underlie ion channel function are permeation/selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labeling/EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. We have focused our attention on a series of prokaryotic K+ and mechanosensitive channels with different energy transduction mechanisms (proton binding, KcsA; membrane stretch, MscL; transmembrane voltage, KvAP). Through site- directed spin labeling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within these channels with high reactivity and specificity. EPR spectroscopy analysis of the spin labeled mutants yields two types of structural information: 1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and 2) distances between pairs of nitroxides through dipole-dipole interactions With a number of these measurements, explicit clues may be gathered for a specific tertiary fold. However, compared to high-resolution X-ray and NMR data, SDSL structural parameters are limited in terms of spatial resolution and abundance/redundancy, reducing the overall models to backbone protein “structures”. Still, EPR-generated models can not only give an idea of the protein global fold but have been instrumental in determining specific functional mechanisms associated with medium-to-large structural rearrangements. Currently, the single, most important challenge to the use of these methodologies continues to be the translation of these clearly informative but limited data sets into reliable and objective structural models. We have pursued partial solutions to this problem, including the use of static reference structures (obtained independently by X-ray analysis), use of distance information for computations based on rigid-body movements, and the use of experimentally-determined solvent accessibilities to calculate global folds and rigid-body rearrangements. Examples of these approaches, applied to ion channel systems will be discussed: proton activation and helix movements in the potassium channel KcsA and the trapping and structural analysis of the open state of MscL, a prokaryotic mechanosensitive channel and the architecture and conformation of the voltage sensor in KvAP, a prokaryotic voltage-dependent potassium channel.

Tea and coffee will be served in R3151 Beckman Institute at 2:30pm.