Membrane-bound conformation of a signal peptide: A transferred nuclear Overhauser effect analysis

Abstract

We have determined the conformation of an analogue of the Escherichia coli LamB signal peptide inserted into a model membrane using the transferred nuclear Overhauser effect (trNOE) NMR technique. In order to make NMR analysis feasible, a water-soluble LamB signal peptide analogue was designed by inserting three basic residues (KRR) into the N-terminal region of the wild-type sequence (with a Val-->Trp mutation for fluorescence measurements), viz., MMITLRKRRKLPLAVAVAAGWMSAQAMA-NH2. For the purpose of the trNOE study, the binding affinity of the peptide for phospholipid vesicles was tuned by adjusting the proportion of acidic lipid in the vesicle. Circular dichroism and fluorescence measurements 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 analysis revealed that the alpha-helix extended from approximately the beginning of the hydrophobic core (residue Leu8) to the C-terminus. The continuity of the helix was somewhat disrupted at the end of the hydrophobic core (around residue Gly17). Furthermore, the topological arrangement of the peptide within the lipid bilayer was explored by NMR line broadening induced by a paramagnetic nitroxide-labeled lipid. The line-broadening results demonstrated that the residues in the helical region are well integrated into the acyl chain region of the bilayer. The N-terminal part of the peptide showed many trNOEs, but without any indication of a helical conformation. The line-broadening analysis indicates that this part of the peptide primarily interacts with the membrane surface.

Phospholipid-induced Monomerization and Signal-peptide-induced Oligomerization of SecA

“…Signal Peptide Synthesis and Purification-The KRR-LamB signal peptide ( ϩ H 3 N-MMITLRKRRKLPLAVAVAAGVMSAQAMA-COO Ϫ ) and the ⌬78 variant ( ϩ H 3 N-MMITLRKRRKLPVAAGVMSAQAMA-C-OO Ϫ ) were synthesized and purified as described (13). The concentration of signal peptide in a concentrated stock solution in water was determined using quantitative amino acid analysis (conducted at the W. M. Keck Biopolymer Facility at Yale University).…”

“…There is substantial evidence that SecA interacts with both the N-terminal signal sequence (6,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) that targets preproteins for export from the cytoplasm as well as the mature region of the preprotein (22,23). These binding interactions presumably allow SecA to transfer polypeptide segments to SecYEG.…”

Use of synthetic signal sequences to explore the protein export machinery

“…We exploited the ability of signal peptides to spontaneously insert into model bilayers in lipid vesicles to determine their membrane-associated conformation, using transferred nuclear Overhauser effect (trNOE) NMR. 25 We also studied the manner in which they associated with the membrane using paramagnetic broadening of the NMR signals arising from the bound conformations, 25 EPR of spin-labeled lipids in the presence of signal peptides, 26 and extensive mapping of fluorescently labeled signal peptides using a variety of exogenous aqueous or lipid-resident quenchers. 20,27,28 The picture that emerged was consistent with all these data: The charged N-terminal region of the signal peptide associates with the lipid head groups of the cis leaflet of the bilayer in an extended conformation, while the hydrophobic core inserts well into the acyl chain region in a helical conformation.…”

Principles of protein folding — A perspective from simple exact models

“…It has been shown that membrane-spanning regions of several integral membrane proteins are helical (Deisenhofer et al, 1985;Yeates et al, 1987). The peptides gramicidin A (Killian, 1992) and Lam B (Wang et al, 1993) undergo a conformational change to an e-helix upon insertion into membranes.…”