Aggregation state of spin-labeled cecropin AD in solution.

A spin-labeled derivative of the ion channel peptide cecropin AD (Fink et al., 1988) was synthesized and used to investigate its aggregation state in water and in the presence of a helix-promoting solvent. A cysteine was introduced at position 33 and spin-labeled using the methanethiosulfonate spin label. In low ionic strength aqueous solution, the peptide is monomeric, and the ESR spectrum indicates a high degree of segmental flexibility at the nitroxide attachment point, consistent with a predominantly random coil conformation. Upon addition of 5-10% (v/v) hexafluoro-2-propanol (HFP), the peptide is induced to aggregate as evidenced by significant motional restriction of the spin label and spin-spin broadening of the ESR lines. At higher concentrations of HFP, the peptide reverts to a monomeric state but retains its folded conformation. Our data suggest that between 5 and 10% HFP the peptide undergoes two structural transitions. The first transition starts at 5% and is very cooperative. Its dependence on ionic strength, temperature, and pH indicates that it involves the interconversion between a random coil and an ordered state stabilized by interpeptide electrostatic and hydrophobic interactions. The second transition, which occurs at 11% v/v HFP, is between the self-associated form and an ordered monomeric form. The analysis of our experimental results demonstrates aggregate formation at 5-10% HFP. This may be relevant to the mechanism of channel formation by cecropins in membranes.