Structure of Tightly Membrane-Bound Mastoparan-X, a G-Protein-Activating Peptide, Determined by Solid-State NMR (original) (raw)
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The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy
Chemistry and Physics of Lipids, 2012
Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.
Solid-state NMR approaches for studying the interaction of peptides and proteins with membranes1
Biochimica Et Biophysica Acta Mr Reviews on Biomembranes, 1998
1. Biochim Biophys Acta. 1998 Nov 10;1376(3):297-318. Solid-state NMR approaches for studying the interaction of peptides and proteins with membranes. Watts A. Biomembrane Structure Unit, Biochemistry Department, Oxford University, Oxford OX1 3QU, UK. awatts@bioch.ox.ac.uk. PMID: 9804977 [PubMed - indexed for MEDLINE]. Publication Types: Research Support, Non-US Gov't; Review. MeSH Terms. Animals; Cytochrome c Group/chemistry; ...
Biophysical Journal, 2014
Phylloseptin-1,-2, and-3 are three members of the family of linear cationic antimicrobial peptides found in tree frogs. The highly homologous peptides encompass 19 amino acids, and only differ in the amino acid composition and charge at the six most carboxy-terminal residues. Here, we investigated how such subtle changes are reflected in their membrane interactions and how these can be correlated to their biological activities. To this end, the three peptides were labeled with stable isotopes, reconstituted into oriented phospholipid bilayers, and their detailed topology determined by a combined approach using 2 H and 15 N solid-state NMR spectroscopy. Although phylloseptin-2 and-3 adopt perfect in-plane alignments, the tilt angle of phylloseptin-1 deviates by 8 probably to assure a more water exposed localization of the lysine-17 side chain. Furthermore, different azimuthal angles are observed, positioning the amphipathic helices of all three peptides with the charged residues well exposed to the water phase. Interestingly, our studies also reveal that two orientation-dependent 2 H quadrupolar splittings from methyl-deuterated alanines and one 15 N amide chemical shift are sufficient to unambiguously determine the topology of phylloseptin-1, where quadrupolar splittings close to the maximum impose the most stringent angular restraints. As a result of these studies, a strategy is proposed where the topology of a peptide structure can be determined accurately from the labeling with 15 N and 2 H isotopes of only a few amino acid residues.
Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These "static" structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD 3-or CF 3-group attached to the C α \C β bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where 15 N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not.
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.
Solid-state NMR structures of integral membrane proteins
Molecular Membrane Biology, 2015
Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid-and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.
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.
Orientations of helical peptides in membrane bilayers by solid state NMR spectroscopy
Solid State Nuclear Magnetic Resonance, 1996
The orientations of helical peptides in membrane bilayers provide important structural information that is directly relevant to their functional roles, both alone and within the context of larger membrane proteins. The orientations can be readily determined with solid state NMR experiments on samples of "N-labeled peptides in lipid bilayers aligned between glass plates. The observed "N chemical shift frequencies can be directly interpreted to indicate whether the peptide's helix axis has a trans-membrane or an in-plane orientation. In order to distinguish between these possibilities on the basis of a single spectral parameter, e.g. the easily measured 15N chemical shift frequency, it is necessary to demonstrate that the secondary structure of the peptide is helical, generally by solution NMR spectroscopy of the same peptide in micelle samples, and that it is immobile in bilayers, generally from solid state NMR spectra of unoriented samples, Six different 20-30 residue peptides are shown to have orientations that fall into the categories of trans-membrane or in-plane helices. A model hydrophobic peptide was found to be trans-membrane, several different amphipathic helical peptides were found to have either trans-membrane or in-plane orientations, and a leader or signal peptide, generally regarded as hydrophobic, was found to have a significant population with an in-plane orientation.
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.