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. Spin labeled gramicidin A (GAsl) was synthesized and studied by ESR, high-field ESR and DQC-ESR in different lipid membranes, microscopically aligned by ISDU and in vesicles. This is an excellent model system for developing our methods for membrane proteins. DQC clearly indicates the presence of pairs in DMPC membranes, as well as membranes build of unsaturated lipids or saturated lipids shorter than DMPC. The interspin distance in pairs (30.9 for DMPC) is in good agreement with our estimates for head-to-head dimers, with small variations, depending on the lipid. Though it is possible that the difference may result from flexibility in the nitroxide tethers, we cannot rule out that the backbone length of the channel dimer may also be lipid-dependent. The analysis of ESR spectra shows for the head-to-head dimer deep embedding and a tilt of the nitroxide group. In the L-beta phase of DPPC and DSPC GAsl takes on a different, apparently double helical, conformation. In this case the nitroxide moiety shows good Z-ordering and is located close to the membrane surface, DQC detects signs of aggregation of more than two molecules. Both DPPC and DSPC in the gel phase have a large hydrophobic mismatch between the dimer length and bilayer thickness, so they apparently do not favor channel formation. Above the L-beta and P-beta phase transition in DPPC double helices transform to channels. The channel formation manifests itself as a disruption of Z-ordering due to the tilt of the nitroxide moiety and a decrease in the polarity of its environment. This conclusion, which could be determined from 9GHz spectra after extensive spectral simulations is very clear at 170GHz even by inspection. The spectral intensity shifts from the Z-region to the XY-region and back by performing a cooling/heating cycle in the aligned DPPC membrane. We attribute the considerable hysteresis in the cycle to a slow dissociation of the channel form. The hysteresis allows us to lock-in the high temperature conformation and to observe the channel formation in the P-beta phase by DQC-ESR.
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