Solid-State NMR Investigation of the Depth of Insertion of Protegrin-1 in Lipid Bilayers Using Paramagnetic Mn2+

Abstract

The depth of insertion of an antimicrobial peptide, protegrin-1 (PG-1), in lipid bilayers is investigated using solid-state NMR. Paramagnetic Mn(2+) ions bind to the surface of lipid bilayers and induce distance-dependent dipolar relaxation of nuclear spins. By comparing the signal dephasing of the peptide with that of the lipids, whose segmental depths of insertion are known, we determined the depths of several residues of PG-1 in 1,2 dilauryl-sn-glycero-3-phosphotidylcholine (DLPC) bilayers. We found that residues G2 at the N-terminus and F12 at the beta-turn of the peptide reside near the membrane surface, whereas L5 and V16 are embedded in the acyl chain region. The depths increase in the order of G2 < F12 < L5 < V16. These intensity-dephasing results are confirmed by direct measurement of the paramagnetically enhanced (13)C transverse relaxation rates. The relative depths indicate that PG-1 is tilted from the bilayer normal, which is consistent with independent solid-state NMR measurements of PG-1 orientation in the same lipids (Yamaguchi et al., 2001). They also indicate that PG-1 is fully immersed in the lipid bilayer. However, a quantitative mismatch between the bilayer thickness and PG-1 length suggests a local thinning of the DLPC bilayer by 8-10 A. The depth sensitivity of this Mn(2+) dephasing technique is tunable with the Mn(2+) concentration to focus on different regions of the lipid bilayer.

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

“…[45;46] Hong et al reported that the paramagnetic quenching can be utilized to measure distances of 13 C sites in membrane bound peptides from the membrane surface with ranging Mn(II) concentration in water phase. [46] Thus, it is probable that signals for residues exposed to the protein surface are selectively quenched by the paramagnetic effects, while the majority of signals for other residues are unaffected. Further studies are needed to examine the possibilities of using the effects for structural analysis.…”

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…47 The two long ends of the elongated molecule experienced stronger T 2 relaxation enhancements, whereas residues in the middle of the hairpin are more shielded from the Mn 2þ effects. Thus, PG-1 spans both leaflets of the DLPC bilayer.…”

“…1(e)]. 47 The former correlates the 1 H signals of dynamic molecules in the membrane sample with 13 C signals of the protein after a 1 H spin diffusion period. The CH 2 and CH 3 signals of the lipid chains are particularly important as the 1 H magnetization source: TM proteins exhibit strong lipid-protein cross peaks in less than 100 ms, whereas surface-bound peptides requires several hundred milliseconds and seconds to develop cross peaks.…”

“…Mn 2þ or other paramagnetic ions bind to the negatively charged phosphate groups on the membrane surface and cannot freely penetrate into the membrane. 47 These paramagnetic ions speed up nuclear spin relaxation in a distance-dependent fashion. Thus, by measuring the PREs of the peptide signals and compare them with those of the lipid carbons, whose depths are well known, one can obtain site-specific depths of the protein residues from the bilayer surface.…”

“…Thus, by measuring the PREs of the peptide signals and compare them with those of the lipid carbons, whose depths are well known, one can obtain site-specific depths of the protein residues from the bilayer surface. 47 Moreover, lipid vesicles for which only one surface of the bilayer is coated with Mn 2þ can be prepared to determine the asymmetry of peptide insertion. 49 For example, with outer-leaflet Mn 2þ -bound samples, peptides that insert only into the outer leaflet of the bilayer will exhibit stronger PRE effects (less residual intensity) than peptides that are distributed into both leaflets of the bilayer.…”