Our current research focuses on bacterial mechanosensitive channels MscL and MscS, which function as osmolyte release valves in prokaryotes. These channels are good models for studies of basic principles of membrane-based sensory mechanisms because they gate directly in response to tension in the lipid bilayer, and their crystal structures are available.
The structural information is used to interpret conductive and gating properties of the channels as a set of distinct protein conformations. Our goals are to identify the intramolecular forces and protein-lipid interactions that define stable conformations and to explain the pathways for gating transitions in such channels.
We use molecular modeling and simulations as a way to formulate a hypothesis, and then rely on a combination of experimental techniques to test it. The work proceeds in cycles as more data is accumulated and as models improve. The patch-clamp characterization of the channels in giant bacterial spheroplasts or liposomes allows estimation of the size of the conducting pores. The kinetic analysis of transitions at different tensions allows us to determine the thermodynamic parameters of gating such as energies, barriers, and spatial scales of the conformational changes between the states. In collaboration with Dr. H. Robert Guy (NIH) we have developed molecular models of different states of MscL based on existing crystal structures. We utilize the power of site-directed mutagenesis, biochemical modifications and cross-linking to test the proximities of certain residues in specific states as predicted by the models. Single-channel recordings combined with disulfide trapping experiments allows us to observe changes in gating caused by engineered disulfide bonds. We have also entered the field of molecular dynamic simulations in hopes to envision the behavior of water, effects of mutations and the detailed pathway for conformational transitions with steered simulations. See the snapshots of our research!
This page was last updated on 01/16/09.