Determination of membrane Peptide orientation by 1H-detected 2H NMR spectroscopy

We demonstrate the application of the proton inverse detected deuteron (PRIDE) NMR technique to the measurement of the orientation of membrane-bound peptides with enhanced sensitivity. Gramicidin D, a transmembrane peptide, and ovispirin, a surface-bound peptide, were used as model systems. The peptides were 2H-labeled by 1H/2H exchange and oriented uniaxially on glass plates. The directly detected 2H spectra of both peptides showed only a strong D(2)O signal and no large quadrupolar splittings. In contrast, the PRIDE spectrum of gramicidin exhibited quadrupolar splittings as large as 281 kHz, consistent with its transmembrane orientation. Moreover, the large D(2)O signal in the directly detected 2H spectra was cleanly suppressed in the PRIDE spectrum. For ovispirin, the 1H indirectly detected 2H spectrum revealed a 104 kHz splitting and a zero-frequency peak. The former reflects the in-plane orientation of most of the helix axis, while the latter results from residues with a magic-angle orientation of the N-D bonds. These are consistent with previous 15N NMR results on ovispirin. The combination of PRIDE and exchange labeling provides an economical and sensitive method of studying membrane peptide orientations in lipid bilayers without the influence of D(2)O and with the ability to detect N-D bonds at the magic angle from the bilayer normal.     

Susceptibility corrections in solid-state NMR experiments with oriented membrane samples. Part I: applications

Chemical shift referencing of solid-state NMR experiments on oriented membranes has to compensate for bulk magnetic susceptibility effects that are associated with the non-spherical sample shape, as described in the accompanying paper [J. Magn. Reson. 164 (2003) 115-127]. The resulting frequency deviations can be on the order of 10 ppm, which is serious for nuclei with a narrow chemical shift anisotropy such as 1H or 13C, and in some cases even 19F. Two referencing schemes are proposed here to compensate for these effects: A flat (0.4 mm) glass container with an isotropic reference molecule dissolved in a thin film of liquid is stacked on top of the oriented membrane sample. Alternatively, the intrinsic proton signal of the hydrated lipid can be used for chemical shift referencing. Further aspects related to magnetic susceptibility are discussed, such as air gaps in susceptibility-matched probeheads, the benefits of shimming, and limitations in the accuracy of orientational constraints. A biological application is illustrated by a series of experiments on the antimicrobial peptide PGLa, aimed at understanding its concentration-dependent membranolytic effect. To address a wide range of molar peptide/lipid ratios between 1:3000 and 1:8, multilayers of hydrated DMPC containing a 19F-labeled peptide were oriented between stacked glass plates. Maintaining an approximately constant amount of peptide gives rise to thick samples (18 plates) at low, and thin samples (3 plates) at high peptide/lipid ratio. Accurate referencing was critical to reveal a small but significant change over 5 ppm in the anisotropic chemical shift of the 19F label on the peptide, indicative of a change in the orientation and/or dynamics of PGLa in the membrane.     

An NMR Investigation of the Conformational Effect of Nitroxide Spin Labels on Ala-Rich Helical Peptides

Nitroxide spin labels, in conjunction with electron spin resonance (ESR) experiments, are extensively employed to probe the structure and dynamics of biomolecules. One of the most ubiquitous spin labeling reagents is the methanethiosulfonate spin label which attaches a spin label selectively to Cys residues via a disulfide bond (Cys-SL). However, the actual effect of the nitroxide spin label upon the conformation of the peptide or protein cannot be unambiguously determined by ESR. In this study, a series of 16-residue Ala-rich helical peptides was characterized by nuclear magnetic resonance techniques. The C_αH chemical shift analysis, NOEs, and³J_NHαcoupling constants for peptides with no Cys, free Cys, and Cys-SL (with the N–O group reduced) were compared. These results indicate that while replacement of an Ala with a Cys residue causes a loss of overall helical structure, the Cys-SL residue is helix supporting, as would be expected for a non-β-branched aliphatic amino acid. Thus, the Cys-SL residue does not perturb helical structure and, instead, exhibits helix-stabilizing characteristics similar to that found for Ala, Met, and Leu.     

Study of the antimicrobial peptide indolicidin and mutants in eukaryotic modelled membrane by molecular dynamics simulations

In this work the interaction of the antimicrobial peptide indolicidin (IND) and its mutants CP10A and CP11 with a eukaryotic membrane model was examined by molecular dynamics simulations. The aim was to analyse the behaviour of these antimicrobial peptides when they interact with a eukaryotic modelled membrane, thereby obtaining atomic detailed observations that are not experimentally available. In the simulations, the widely studied dipalmitoylphosphatidylcholine hydrated bilayer was used as a eukaryotic membrane model. In agreement with experimental observations, the peptides IND, CP10A, and CP11 insert into the bilayer differently; the peptides that insert more deeply present the major hemolytic activities. The hydrophobic residues are responsible for the insertion, but some Trp residues of the peptides remain at the bilayer/water interface because they interact with the bilayer choline groups by cation-π interactions that should be important for recognition of eukaryotic membrane by the three studied peptides.     

新疆家蚕抗菌肽Cecropin-XJ与细菌DNA相互作用的光谱研究

抗菌肽的抗菌机理研究主要集中在抗菌肽与细菌细胞膜作用方面,抗菌肽是否与细菌的染色体DNA作用尚不清楚。为了探讨新疆家蚕抗菌肽Cecropin-XJ抗细菌的作用机理,利用紫外光谱及以溴化乙锭(EthidiumBromide,EB)为荧光探针的荧光光谱方法研究抗菌肽Cecropin-XJ与金黄色葡萄球菌DNA在体外的相互作用,计算获得抗菌肽与DNA的结合常数和成键位点数。结果显示,抗菌肽使DNA发生了明显的增色效应,并使DNA的荧光强度增强,抗菌肽能与EB竞争性的结合DNA,表明抗菌肽可能与DNA双螺旋的沟槽结合;在抗菌肽存在下,DNA与EB作用的结合常数和成键位点数都发生变化,表明抗菌肽以嵌入和非嵌入两种方式与DNA相互作用。文章从分子水平上初步阐述了抗菌肽与细菌DNA的作用模式和结构特征,为深入研究抗菌肽的作用机理奠定了基础。     

Anisotropic orientation of the carbon fiber/liquid crystalline epoxy resin composites

Anisotropic orientation of carbon fiber (CF)/liquid crystalline epoxy (LCE) resin composite was readily induced during curing on a CF surface along a long molecular axis of CF. Orientation of LCE was confirmed with polarized optical microscope (POM) and wide angle X-ray diffractometer (WAXD). In addition, anisotropic ordering of LCE was correlated with curing rate, dynamic mechanical properties and thermal expansion behaviors of CF/LCE composite. Curing of LCE was accelerated in the presence of CF and the rubbery modulus of the CF/LCE composites cured at low temperature was enhanced by long-range, long axis orientational ordering of the LCE resin along a CF surface. Fully cured CF/LCE composite showed a negative coefficient of thermal expansion in the fiber direction. These results obtained in this study are interpreted in terms of structural changes occurring during curing.     

Potential energy surface of alanine polypeptide chains

The multidimensional potential energy surfaces of the peptide chains consisting of three and six alanine (Ala) residues have been studied with respect to the degrees of freedom related to the twist of these molecules relative to the peptide backbone (these degrees of freedom are responsible for the folding of such peptide molecules and proteins). The potential energy surfaces have been calculated ab initio within the framework of the density functional theory taking into account all electrons in the system. The probabilities of transitions between various stable conformations of polypeptide molecules are evaluated. The results are compared to the data obtained by molecular dynamics simulations and to the available experimental data. The influence of the secondary structure of the polypeptide chain on its conformational properties with respect to rotations has been studied. It is shown that, in a chain of six amino acid (Ala) residues, the secondary structure type (helix or sheet conformation) influences the stable isomer states of the polypeptide.     

Interactions between oligopeptides and oxidised titanium surfaces detected by vibrational spectroscopy

Five alternating polar/hydrophobic oligopeptides derived from EAK 16 (AEAEAKAKAEAEAKAK) were examined in comparison with EAK 16 (peptide 1) both after solubilisation/lyophilisation and deposition on oxidised titanium surfaces. The peptides were synthesised for their possible use as biomimetic materials due to their self‐assembling properties and the presence, in one of them, of the arginine‐glycine‐aspartic (RGD) sequence, an active modulator of cell adhesion. Infrared (IR) and Raman spectroscopies were used to investigate the influence of the amino acid substitution on the self‐assembling properties of the peptides under both experimental conditions. In the lyophilised peptides, β‐sheet was the prevailing conformation (65–69%) as in EAK 16, irrespective of acid substitution (E→D, peptide 2), basic substitution (K→O, peptide 3), hydrophobic spacer substitution (A→Abu, peptide 4 and A→Y, peptide 5) and RGD insertion (peptide 6). After deposition on oxidised titanium, the main conformation remained β‐sheet. The side‐chain shortening of the acidic amino acid residue (peptide 2) or the insertion of a rigid and bulky residue such as Y (peptide 5) decreased the self‐assembling ability more than the side‐chain shortening of the basic amino acid residue (peptide 3) or the insertion of the RGD head (peptide 6). The interaction with the oxidised titanium surface was mainly due to carboxylate groups with a bidentate bridging coordination and C  O peptidic groups. Copyright © 2010 John Wiley & Sons, Ltd.     

Dynamic aspect of bacteriorhodopsin as a typical membrane protein as revealed by site-directed solid-state 13C NMR

We demonstrate here a general feature of dynamic aspect of membrane proteins as revealed by site-directed 13C NMR studies on bacteriorhodopsin (bR) as a typical membrane protein and a variety of mutants at ambient temperature. 13C NMR signals of [3-13C]Ala- or [1-13C]Val-labeled proteins were assigned regio-specifically with reference to the data of the conformation-dependent 13C chemical shifts from model polypeptides, followed by site-specific assignment based on site-directed mutants. Revealed picture of membrane protein at ambient temperature is not static in contrast to anticipation from crystalline structures but flexible enough to undergo a variety of local fluctuations with frequencies from 10(2) to 10(8)Hz, as pointed out already. This picture was further refined by taking into account of residue-specific dynamics of interfacial domains between the surface and inner part of the transmembrane helices and conformational fluctuation induced by the presence of a kinked structure. The residue-specific dynamics of the former was revealed by observation of broadened or suppressed peaks from the interfacial domains caused by acquisition of internal fluctuation motions interfered with frequencies of proton decoupling or magic angle spinning. The presence of such suppressed peaks due to molecular fluctuations in the interfacial domains was further confirmed by insensitivity of the peak-intensities from the interfacial domains in spite of the presence of accelerated relaxation rate to nearby residues from surface bound Mn2+ ion. Further, conformational change of the transmembrane alpha-helix F due to a plausible kinked structure at Pro 186 was confirmed in view of specific displacements of Ala 184 and Val 187 13C NMR peaks from chemically synthesized [3-13C]Ala(184)-, [1-13C]Val(187)-labeled wild type and P186L mutant of transmembrane fragment F(164-194) incorporated into lipid bilayer. It is emphasized that the observed displacement of [3-13C]-labeled Ala 184 peak at 17.4 ppm in the presence of kinked structure in this model peptide is consistent with that of intact protein at 17.27 ppm.     

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

We have shown that bicelles prepared from dilauryl phosphatidylcholine (DLPC) and dipalmitoyl phosphatidylcholine (DPPC) align in a magnetic field under conditions similar to the more common dimyristoyl phosphatidylcholine (DMPC) bicelles. In addition, a model transmembrane peptide, P16, with a hydrophobic stretch of 24 A, and specific alanine-d(3) labels, was incorporated into all of the different bicelles. The long-chain phospholipid (DLPC, DMPC, or DPPC) remained unperturbed upon incorporation of the peptide while the quadrupolar splitting of the short-chain phospholipid along the bicelle rim increased by varying degrees in the different bicelle systems. The change in quadrupolar splitting of the short-chain phospholipids was attributed to changes in either fluidity of the planar region of the bicelle or differences in overall lipid packing. When the hydrophobic stretch of the bilayer was 22.8 (DMPC) or 26.3 A (DPPC), the peptide tilt was found to be transmembrane (33-35 degrees with respect to the bicelle normal). When the hydrophobic stretch of the bilayer was 19.5 A (DLPC), the peptide quadrupolar splittings suggested a loss of transmembrane orientation. When tryptophan was incorporated in the middle of the transmembrane region, the transmembrane orientation was also lost.     

Interactions of lactoferricin B derivatives with model cell membrane studied by Raman spectroscopy

In this work, we employed Raman spectroscopy to study the effect of the antimicrobial peptide lactoferricin B (LfB) on model cell membranes. We used two derivatives of LfB (RRWQWRMKKLG and RRWQWR) with broad‐spectrum activity against gram‐positive and gram‐negative bacteria, fungus, viruses and tumors. Raman spectra of the peptides showed no conformational change in the temperature range 4–60 °C. The positions of the amide I and amide III bands suggest that in an aqueous solution these peptides preferentially adopt a random coil‐like conformation. We also investigated the effect the peptides had on the melting behavior of model cell membranes composed of zwitterionic lipid dipalmitoylglycero‐phosphocholine (DPPC) and anionic lipid dipalmitoylglycero‐phosphoglycerol (DPPG). Raman CH stretching bands were used to follow the melting of the lipid vesicles. We found that the melting of DPPC lipid vesicles is not affected by the presence of the peptides, while the presence of the peptides reduced cooperativity of the phase transition for anionic DPPG vesicle, suggesting that both peptides interact strongly and specifically with this model cell membrane composed of anionic lipid. Copyright © 2006 John Wiley & Sons, Ltd.     

Dynamics of alpha helix formation in the CSAW model

We study the folding dynamics of polyalanine (Ala20), a protein fragment with 20 residues whose native state is a single alpha helix. We use the CSAW model (conditioned self-avoiding walk), which treats the protein molecule as a chain in Brownian motion, with interactions that include hydrophobic force and internal hydrogen bonding. We find that large-scale structures form before small-scale structures, and obtain the relevant relaxation times. We find that helix nucleation occurs at two separate points on the protein chain, one near each end. The evolution of small- and large-scale structures involves different mechanisms. While the former can be described by rate equations that govern the growth of helical content, the latter is akin to the relaxation of an elastic solid.     

Orientation of TOAC amino-acid spin labels in alpha-helices and beta-strands

The orientation of alpha-helices or beta-strands, e.g., in membranes, can be determined from EPR order parameters of (2,2,6,6-tetramethyl-piperidine-1-oxy-4-amino-4-carboxylic acid) TOAC amino-acid spin labels incorporated in the polypeptide backbone. This requires knowledge of the inclination of the nitroxide axes, relative to the alpha-helix or beta-strand axis. Crystal structures of TOAC-containing peptides are used to derive the spin-label orientation relative to refined alpha-poly-l-alanine and beta-poly-l-alanine structures. The spin-label z-axes of the two mirror-image TOAC twist-boat conformers are inclined at 13+/-2 degrees and 65+/-3 degrees , respectively, to the alpha-helix axis, or at 25+/-3 degrees and 32+/-3 degrees to the beta-strand axis.     

UV resonance Raman spectroscopy probes the localization of tryptophan‐containing antimicrobial peptides in lipid vesicles

In this work we employed UV resonance Raman spectroscopy with 229 nm excitation to study two tryptophan‐containing antimicrobial peptides with a broad‐spectrum activity against Gram‐positive and Gram‐negative bacteria: lactoferricin B (LfB, RRWQWRMKKLG) and pEM‐2 (KKWRWWLKALAKK). UV resonance Raman spectra of both peptides are dominated by tryptophan bands. Raman spectra of LfB and pEM‐2 in D₂O and 2,2,2‐trifluoro ethanol (TFE) have been measured and used to identify the hydrogen‐bond strength marker bands W6 and W17. The tryptophan doublet, W7, at 1340 and 1360 cm⁻¹ was used to detect an increase in the hydrophobicity of Trp environment in TFE. The spectra of LfB in complex with model cell membranes composed of zwitterionic dipalmitoylglycero‐phosphocholine (DPPC) or anionic dipalmitoyglycero‐phosphoglycerol (DPPG) lipid vesicles revealed a more hydrophobic Trp environment in DPPG, suggesting stronger interactions between the cationic peptide and anionic model cell membrane. Copyright © 2008 John Wiley & Sons, Ltd.     

Fluorescent Temporin B Derivative and its Binding to Liposomes

Temporins are short (10–13 amino acids) and linear antimicrobial peptides first isolated from the skin of the European red frog, Rana temporaria, and are effective against Gram-positive bacteria and Candida albicans. Similarly to other antimicrobial peptides, the association of temporins to lipid membranes has been concluded to underlie their antimicrobial effects. Accordingly, a detailed understanding of their interactions with phospholipids is needed. We conjugated a fluorophore (Texas Red) to a Cys containing derivative of temporin B (temB) and investigated its binding to liposomes by fluorescence spectroscopy. Circular dichroic spectra for the Cys-mutant recorded in the absence and in the presence of phospholipids were essentially similar to those for temB. A blue shift in the emission spectra and diminished quenching by ferrocyanide (FCN) of Texas Red labeled temporin B (TRC-temB) were seen in the presence of liposomes. Both of these changes can be attributed to the insertion of the Texas Red into the hydrophobic region of the bilayer. Resonance energy transfer, steady state anisotropy, and fluorescence lifetimes further demonstrate the interaction of TRC-temB with liposomes to be enhanced by negatively charged phospholipids. Instead, cholesterol attenuates the association of TRC-temB with membranes. The interactions between TRC-temB and liposomes of varying negative surface charge are driven by electrostatics as well as hydrophobicity. Similarly to native temporin B also TRC-temB forms amyloid type fibers in the presence of negatively charged liposomes. This property is likely to relate to the cytotoxic activity of this peptide.     

Beyond NMR spectra of antimicrobial peptides: Dynamical images at atomic resolution and functional insights

There is a considerable current interest in understanding the function of antimicrobial peptides for the development of potent novel antibiotic compounds with a very high selectivity. Since their interaction with the cell membrane is the major driving force for their function, solid-state NMR spectroscopy is the unique method of choice to study these insoluble, non-crystalline, membrane-peptide complexes. Here I discuss solid-state NMR studies of antimicrobial peptides that have reported high-resolution structure, dynamics, orientation, and oligomeric states of antimicrobial peptides in a membrane environment, and also address important questions about the mechanism of action at atomic-level resolution. Increasing number of solid-state NMR applications to antimicrobial peptides are expected in the near future, as these compounds are promising candidates to overcome ever-increasing antibiotic resistance problem and are well suited for the development and applications of solid-state NMR techniques.     

Solid state NMR analysis of the dipolar couplings within and between distant CF3-groups in a membrane-bound peptide

Dipolar couplings contain information on internuclear distances as well as orientational constraints. To characterize the structure of the antimicrobial peptide gramicidin S when bound to model membranes, two rigid 4-CF3-phenylglycine labels were attached to the cyclic backbone such that they reflect the behavior of the entire peptide. By solid state 19F NMR we measured the homonuclear dipolar couplings of the two trifluoromethyl-groups in oriented membrane samples. Using the CPMG experiment, both the strong couplings within each CF3-group as well as the weak coupling between the two CF3-groups could be detected. An intra-CF3-group dipolar coupling of 86 Hz and a weak inter-group coupling of 20 Hz were obtained by lineshape simulation of the complex dipolar spectrum. It is thus possible to explore the large distance range provided by 19F-labels and to resolve weak dipolar couplings even in the presence of strong intra-CF3 couplings. We applied this approach to distinguish and assign two epimers of the labeled gramicidin S peptide on the basis of their distinct 19F dipolar coupling patterns.     

Vibrational spectroscopy and DFT calculations of di‐amino acid cyclic peptides. Part I: cyclo(Gly‐Gly), cyclo(L‐Ala‐L‐Ala) and cyclo(L‐Ala‐Gly) in the solid state and in aqueous solution

Investigations of the vibrational spectra of cyclo(Gly‐Gly), cyclo(L‐Ala‐L ‐Ala) and cyclo(L ‐Ala‐Gly) are reported. Raman scattering and Fourier transform infrared (FTIR) spectra of solid‐state and aqueous protonated samples, as well as their corresponding N‐deuterated isotopomers, have been examined. In addition, density functional theory (DFT) (B3‐LYP/cc‐pVDZ) calculations of molecular structures and their associated vibrational modes were carried out. In each case, the calculated structures of lowest energy for the isolated gas‐phase molecules have boat conformations. Assignments have been made for the observed Raman and FTIR vibrational bands of the cyclic di‐amino acid peptides (CDAPs) examined. Raman polarization studies of aqueous phase samples are consistent with C₂ and C₁ symmetries for the six‐membered rings of cyclo(L‐Ala‐L‐Ala) and cyclo(L‐Ala‐Gly), respectively. There is a good correlation between experimental and calculated vibrational bands for the three CDAPs. These data are in keeping with boat conformations for cyclo(L‐Ala‐L‐Ala) and cyclo(L‐Ala‐Gly) molecules, predicted by the ab initio calculations, in both the solid and aqueous solution states. However, Raman spectroscopic results might infer that cyclo(L‐Ala‐Gly) deviates only slightly from planarity in the solid state. The potential energy distributions of the amide I and II modes of a cis‐peptide linkage are shown to be significantly different from those of the trans‐peptides. For example, deuterium shifts have shown that the cis‐amide I vibrations found in cyclo(Gly‐Gly), cyclo(L‐Ala‐L‐Ala), and cyclo(L‐Ala‐Gly) have larger N‐H contributions compared to their trans‐amide counterparts. Compared to trans‐amide II vibrations, cis‐amide II vibrations show a considerable decrease in N H character. Copyright © 2009 John Wiley & Sons, Ltd.     

Asymmetry of calmodulin revealed by peptide binding

The binding of amphiphilic peptides to calmodulin has been studied using fluorescence energy transfer techniques. Calmodulin has no tryptophan residues but possesses two tyrosines (at positions 99 and 138) in the C-terminal half of the protein. The peptides have a single tryptophan which serves as energy acceptor for the protein tyrosine fluorescence. For the binding of mastoparan or peptide Baa17, with a tryptophan at position 3, the observed quenching of the tyrosine fluorescence of over a factor of 2 corresponds to an average tyrosine-trytophan distance of less than 14 Å. These results indicate that the peptides binds preferentially with the tryptophan in the C-terminal half of the protein.     

Probing membrane surfaces and the location of membrane-embedded peptides by (13)C MAS NMR using lanthanide ions

A simple but efficient (13)C MAS NMR method is presented for the determination of the location of embedded molecules such as peptides relative to biological membrane surfaces by exploiting the interaction with paramagnetic lanthanide ions. Using various aqueous Dy(3+) concentrations a distance-dependent differential paramagnetic quenching of NMR lipid resonance intensities for specific carbon sites was observed, with residues at the bilayer surface quenched effectively and hydrophobic sites unaffected by Dy(3+). Tested on the membrane-embedded 50 residue long M13 coat protein, (13)C labeled at its Val-29 and Val-31 residues, no paramagnetic quenching was observed for the peptide resonances by Dy(3+), suggesting that Val-29 and Val-31 are not in close proximity to the bilayer interface, but buried deeply inside the hydrophobic region of the lipid bilayer.