PhoE signal peptide inserts into micelles as a dynamic helix-break-helix structure, which is modulated by the environment. A two-dimensional1H NMR study

Proteins that are destined for export out of the cytoplasm of Escherichia coli cells are synthesized as precursor proteins with N-terminal extensions or signal sequences, which are essential for translocation of the protein across the inner membrane. Signal sequences contain very little primary sequence homology, and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of signal sequences, we have studied the signal peptide of PhoE (MKKSTLALVVMGIVASASVQA) by two-dimensional nuclear magnetic resonance and circular dichroism in different membrane mimetic environments. The secondary structure of the PhoE signal peptide was analyzed via interresidue nuclear Overhauser enhancement measurements, chemical shifts of backbone protons, and by measuring amide proton exchange. The membrane mimetic environments studied were trifluoroethanol (TFE) and micelles of sodium dodecyl sulfate (SDS) or dodecylphosphocholine (DPC). In all systems α-helix formation was observed. In TFE, the α-helix stretches from the positively charged N-terminus to Ser18s. In SDS and DPC micelles, the N- and C-terminal α-helical half are separated from each other by a kink at the Gly12position, with the helical content being higher at the N-terminus and lower at the C-terminus. In zwitterionic DPC micelles, the C-terminal region has a less regular or more flexible structure compared to SDS. The insertion of the PhoE signal peptide into the hydrophobic environment of the micelles was demonstrated by the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances. It is proposed that conformational rearrangements at Gly12induced by the lipid environment play a role in the export process of the precursor protein.