Spin-labeled gramicidin a: channel formation and dissociation.
Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14583-1301, USA.
Boris G
BG
Dzikovski
Petr P
PP
Borbat
Jack H
JH
Freed
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006096:Gramicidin / Q000737:chemistry*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D008567:Membranes, Artificial; D044367:Phase Transition; D016062:Porosity; D011487:Protein Conformation; D013113:Spin Labels
D007473:Ion Channels; D008563:Membrane Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D013113:Spin Labels; D006096:Gramicidin