MEMBRANE-BOUND CONFORMATION OF A SIGNAL PEPTIDE - A TRANSFERRED NUCLEAR OVERHAUSER EFFECT ANALYSIS

We have determined the conformation of an analogue of the Escherichia coli LamB signal peptide inserted into a model membrane using the tran sferred nuclear Overhauser effect (trNOE) NMR technique. In order to m ake NMR analysis feasible, a water-soluble LamB signal peptide analogu e was designed by inserting three basic residues (KRR) into the N-term inal region of the wild-type sequence (with a Val --> Trp mutation for fluorescence measurements), viz., MMITLRKRRKLPLAVAVAAGWM-SAQAMA-NH2. For the purpose of the trNOE study, the binding affinity of the peptid e for phospholipid vesicles was tuned by adjusting the proportion of a cidic lipid in the vesicle. Circular dichroism and fluorescence measur ements showed that the KRR-LamB signal peptide spontaneously inserted into the lipid bilayer with a conformational transition from a mostly random coil to a predominantly alpha-helical structure. The trNOE anal ysis revealed that the alpha-helix extended from approximately the beg inning of the hydrophobic core (residue Leu8) to the C-terminus. The c ontinuity of the helix was somewhat disrupted at the end of the hydrop hobic core (around residue Gly17). Furthermore, the topological arrang ement of the peptide within the lipid bilayer was explored by NMR line broadening induced by a paramagnetic nitroxide-labeled lipid. The lin e-broadening results demonstrated that the residues in the helical reg ion are well integrated into the acyl chain region of the bilayer. The N-terminal part of the peptide showed many trNOEs, but without any in dication of a helical conformation. The line-broadening analysis indic ates that this part of the peptide primarily interacts with the membra ne surface.