The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy (original) (raw)
Related papers
2007
Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the (15)N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the (2)H solid-state NMR spectra acquired from peptides labelled with 3,3,3-(2)H(3)-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C(alpha)-C(beta) bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu(1-27) and Influenza A M2(22-46) are shown. Whereas the (15)N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.
Biophysical Journal, 2011
The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2 H-alanine quadrupolar splittings together with 15 N/ 1 H dipolar couplings and 15 N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA) 6 LWLAGA-amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5 -35 in lipid bilayer membranes. By comparing individual and combined analyses of specifically 2 H-or 15 N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<~10 ) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional 15 N as well as 2 H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with 2 H or 15 N data alone. Importantly, for peptides that tilt significantly more than 10 from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Solid-state NMR spectroscopy is being developed at a fast pace for the structural investigation of immobilized and non-crystalline biomolecules. These include proteins and peptides associated with phospholipid bilayers. In contrast to solution NMR spectroscopy, where complete or almost complete averaging leads to isotropic values, the anisotropic character of nuclear interactions is apparent in solid-state NMR spectra. In static samples the orientation dependence of chemical shift, dipolar or quadrupolar interactions, therefore, provides angular constraints when the polypeptides have been reconstituted into oriented membranes. Furthermore, solid-state NMR spectroscopy of aligned samples offers distinct advantages in allowing access to dynamic processes such as topological equilibria or rotational diffusion in membrane environments. Alternatively, magic angle sample spinning (MAS) results in highly resolved NMR spectra, provided that the sample is sufficiently homogenous. MAS spinning solid-state NMR spectra allow to measure distances and dihedral angles with high accuracy. The technique has recently been developed to selectively establish through-space and through-bond correlations between nuclei, similar to the approaches well-established in solution-NMR spectroscopy. D
Biochemistry, 2004
Knowledge of the alignment of R-helical polypeptides with respect to the membrane surface and their dynamics in the membrane are key to understanding the functional mechanisms of channels, antibiotics, and signal or translocation peptides. In this paper polypeptides have been labeled with [3,3,3-2 H 3 ]alanine as well as with 15 N at single site amide positions and reconstituted into oriented phospholipid bilayers. A transmembrane and two amphipathic helical polypeptides with the deuterium label at orthogonal positions have been investigated by deuterium and proton-decoupled 15 N solid-state NMR spectroscopy. The 15 N chemical shift measurements and the deuterium quadrupole splitting exhibit a highly complementary functional dependence with respect to the spatial alignment of the polypeptide. Therefore, the combination of these two measurements allows one to determine both the tilt and the rotational pitch angle with high precision. In addition, the deuterium line shape is very sensitive to mosaic spread and the relative orientation of the peptide. The solid-state NMR measurements indicate that the model sequences exhibit a small degree of mosaicity, when at the same time the phospholipid headgroup region is significantly distorted. Furthermore, the 2 H solid-state NMR spectra reveal small orientational and dynamic differences when the fatty acyl chain composition of the phosphatidylcholine bilayers is modified.
Concepts and Methods of Solid-State NMR Spectroscopy Applied to Biomembranes
Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this paper. Membrane lipids with 2 H-labeled acyl chains or polar head groups are studied using 2 H NMR to yield knowledge of their atomistic structures in relation to equilibrium properties. This review demonstrates the principles and applications of solid-state NMR by unifying dipolar and quadrupolar interactions and highlights the unique features offered by solid-state 2 H NMR with experimental illustrations. For randomly oriented multilamellar lipids or aligned membranes, solid-state 2 H NMR enables direct measurement of residual quadrupolar couplings (RQCs) due to individual C− 2 H-labeled segments. The distribution of RQC values gives nearly complete profiles of the segmental order parameters S CD (i) as a function of acyl segment position (i). Alternatively, one can measure residual dipolar couplings (RDCs) for natural abundance lipid samples to obtain segmental S CH order parameters. A theoretical mean-torque model provides acyl-packing profiles representing the cumulative chain extension along the normal to the aqueous interface. Equilibrium structural properties of fluid bilayers and various thermodynamic quantities can then be calculated, which describe the interactions with cholesterol, detergents, peptides, and integral membrane proteins and formation of lipid rafts. One can also obtain direct information for membrane-bound peptides or proteins by measuring RDCs using magic-angle spinning (MAS) in combination with dipolar recoupling methods. Solid-state NMR methods have been extensively applied to characterize model membranes and membrane-bound peptides and proteins, giving unique information on their conformations, orientations, and interactions in the natural liquid-crystalline state. CONTENTS
Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers
Journal of Biomolecular NMR, 1995
A three-dimensional 1 H chemical shift/ 1 H-15 N dipolar coupling/ 15 N chemical shift correlation spectrum was obtained on a sample of specifically 15 N-labeled magainin peptides oriented in lipid bilayers between glass plates in a flat-coil probe. The spectrum showed complete resolution of the resonances from two labeled amide sites in all three dimensions. The three orientationally dependent frequencies associated with each resonance enabled the orientation of the peptide planes to be determined relative to the direction of the applied magnetic field. These results demonstrate the feasibility of multiple-pulse spectroscopy in a flat-coil probe, the ability to measure three spectral parameters from each site in a single experiment, and the potential for resolving among many labeled sites in oriented membrane proteins.
Progression of NMR studies of membrane-active peptides from lipid bilayers to live cells
Journal of magnetic resonance (San Diego, Calif. : 1997), 2015
Understanding the structure of membrane-active peptides faces many challenges associated with the development of appropriate model membrane systems as the peptide structure depends strongly on the lipid environment. This perspective provides a brief overview of the approach taken to study antimicrobial and amyloid peptides in phospholipid bilayers using oriented bilayers and magic angle spinning techniques. In particular, Boltzmann statistics REDOR and maximum entropy analysis of spinning side bands are used to analyse systems where multiple states of peptide or lipid molecules may co-exist. We propose that in future, rather than model membranes, structural studies in whole cells are feasible.
Biochimie, 2009
The 2 H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The 2 H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.
On the Role of NMR Spectroscopy for Characterization of Antimicrobial Peptides
Membrane Proteins, 2013
Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structurefunction paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based soley on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid-and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs.
Journal of the American Chemical Society, 2008
Characterization of the oligomerization of membrane-associated peptides is important to understand the folding and function of biomolecules like antimicrobial peptides, fusion peptides, amyloid peptides, toxins, and ion channels. However, this has been considered to be very difficult, because the amphipathic properties of the constituents of the cell membrane pose tremendous challenges to most commonly used biophysical techniques. In this study, we present the application of a simple 14 N solidstate NMR spectroscopy of aligned model membranes containing a phosphatidyl choline lipid to investigate the oligomerization of membrane-associated peptides. Since the near-symmetric nature of the choline headgroup of a phosphocholine lipid considerably reduces the 14 N quadrupole coupling, there are significant practical advantages in using 14 N solid-state NMR experiments to probe the interaction of peptide or protein with the surface of model membranes. Experimental results for several membrane-associated peptides are presented in this paper. Our results suggest that the experimentally measured 14 N quadrupole splitting of the lipid depends on the peptide-induced changes in the electrostatic potential of the lipid bilayer surface and therefore on the nature of the peptide, peptide-membrane interaction, and peptide-peptide interaction. It is inferred that the membrane orientation and oligomerization of the membrane-associated peptides can be measured using 14 N solid-state NMR spectroscopy.