Conformational Plasticity of the Cell-Penetrating Peptide SAP As Revealed by Solid-State F-NMR and Circular Dichroism Spectroscopies.

The cell-penetrating peptide SAP, which was designed as an amphipathic poly-l-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state F-NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. Trifluoromethyl-bicyclopentyl-glycine (CF-Bpg) and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF-MePro) were used as labels for F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF-MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered "raft"-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a nonaggregated state was structured as a mixture of nonideal extended conformations reflecting the equilibrium already present in solution, i.e., before binding to the membrane.