Non-channel forms of gramicidin in the lipid membrane by double quantum coherence ESR
In previous work on channel formation using spin labeled Gramicidin A (GAsl) we found that the non-channel form prevails in the gel phase of mismatching lipids (DPPC, DSPC). We have now addressed the assignment of non-channel forms and studied the complex equilibrium in the gramicidin/lipid system, which includes channels (i.e. head-to-head dimers [HHD]), non-channel dimers, and monomers. To study non-channel forms of spin labeled GA, which can aggregate, we labeled two different sites on the same gramicidin molecule, instead of measuring the distance between spin labels attached at the same position on both dimer-forming molecules. This intramolecular distance between the spin labels could be used as a fingerprint of the particular conformation. Interactions between spin labels on different gramicidin molecules were eliminated by substantially diluting the double spin-labeled gramicidin by unlabeled gramicidin. Several new spin labeled gramicidin compounds were synthesized and used. In octanol, gramicidin exists mainly as a double helical (DH) ππ5.6 left-handed antiparallel dimer, and we determined interspin distances for this structure. GAD with both N- and C-termini labeled and GALN, single-labeled at the N-terminus, substantially impair their propensity to HHD formation. In most lipids we observed two different conformations of GAD. The interspin distance in the main fraction (20.0Å) is consistent with the monomeric state. Double helices (31.6Å, exactly as in octanol) are a minor component with the fraction changing in the saturated lipid series in the following order: DLPC£DMPC<DSPC<DPPC. We also showed by DQC that both gramicidin monomers and DH exist in all studied lipid phases only in aggregates.
Based on interspin distances and aggregation behavior monitored by spin-labeling DQC- and CW-ESR we conclude:
- The equilibrium responsible for channel formation/dissociation in gramicidin/lipid systems is between HHD and aggregates of monomers.
- The only form of gramicidin which exists as distinct entities is HHD. In any lipid phase DH’s and monomers aggregate.
- The monomers are similar to a single gramicidin of a HHD, having the same backbone structure but a wider variety of conformations for the sidechains.
- DH dimers are present as a minor (and likely) metastable fraction.
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