Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

Abstract

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed.

Interaction of mastoparan with membranes studied by 1H‐NMR spectroscopy in detergent micelles and by solid‐state 2H‐NMR and 15N‐NMR spectroscopy in oriented lipid bilayers

“…The latter lineshape also confirms that the central dip of Fig. 6 spectrum c is not an artefact arising from inefficient cross‐polarization as mentioned in other 15 N‐NMR studies of peptides [33]. Secondly, the oriented lineshape (Fig.…”

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers

“…Similarly, when the signal sequence of the M13 coat protein was studied in lipid bilayers by 15 N-NMR, the peptide was found to lie predominantly in-plane but with a very broad orientational distribution [33]. The mastoparan±lipid interaction has been interpreted in terms of a pore-forming model, based on the dose±response curve of peptide-induced ion permeation [14].…”

Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence

“…Peptide-lipid interactions can affect both protein and bilayer structure. Previous studies have suggested that these interactions can serve several regulatory roles such as controlling the membrane-association of lytic peptides [1], modulating membrane-protein activity [2], promoting peptide aggregation [3][4][5][6][7], driving the formation of lipid microdomains, inducing segregation of proteins within the membrane [8][9][10], determining protein sorting within the secretion pathway [11,12], and allowing for cell membrane recognition or fusion [13][14][15]. Previous studies of model transmembrane peptides comprised of polyleucine or Ala-Leu repeats of varying lengths in model membrane systems have shown they form stable a-helices [11,[16][17][18][19][20][21][22][23][24].…”