Solid-state NMR experiments will be performed on oriented membranes containing modified, labeled voltage-dependent gramicidin channels at both zero and nonzero voltage. Systems for study will include channels in which an asymmetry is introduced near the channel center, by modifying only one of the subunits, and a newly discovered homodimeric voltage-dependent gramicidin channel. The modified channels show nonlinear behavior and voltage-dependent gating among two or more conducting states. The helical subunits are held together by hydrogen bonds that link amino acids 1-5 of each subunit, and these regions of sequence are responsible for the gating. Specific backbone and side chain groups within the first five residues of each subunit will be selectively labeled with 2H or l5N by solid-phase chemical synthesis. Solid-state NMR spectroscopy will be used to characterize the average orientations and dynamics of specifically labeled sites that sense voltage changes in lipid bilayers. The homodimeric samples will be advantageous because all of their channels will have the same chemical composition. This feature will allow studies at nonzero potential to be feasible for the first time.
Lay: This project seeks to define a molecular mechanism for the voltage-dependent gating of ion-selective membrane channels using the only known gated system for which the chemical architecture is understood at the molecular level. The model system for study consists of several recently discovered chemically modified derivatives of the antibiotic gramicidin A. These derivatives were designed to have voltage-dependent properties that would help further the basic understanding of channel gating. Solid-state magnetic resonance experiments will be performed on these designed gramicidin channels supported within oriented membranes at zero voltage, and in the presence of a pulsing electric field. The results will be important for understanding the chemical and molecular basis for channel gating in biological membranes.
