Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR.

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.