Solid-state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana

Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335–365) contained the putative membrane anchor of the protein (residues 339–357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly ¹³C- and ¹⁵N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. ³¹P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the ²H NMR spectra of the POPC in the membrane. The conformation of TWD1(335–365) was investigated by ¹³C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of −30°C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven ¹³C spin diffusion and ¹³C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular α-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static ¹⁵N NMR spectra of TWD1(335–365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335–365) adopts an orientation perpendicular to the membrane normal. At 30°C, the peptide undergoes intermediate time scale motions. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

A compound representing the D-glycerate terminus of the methylglucose-containing polysaccharide of Mycobacterium smegmatis.

In order to study the structure of the methylglucose-containing polysaccharide (MGP) of Mycobacterium smegmatis by NMR spectroscopy, we have prepared the model compound O-alpha-D-glucopyranosyl-(1 leads to 2)-D-glyceric acid. This compound, which represents the aglycon-containing terminus of MGP, was made from leucorse [O-alpha-D-glucopyranosyl-(1 leads to 5)-D-fructopyranose] by successive treatment with sodium borohydride, lead tetraacetate, and hypobromite. The structure of O-alpha-D-glucopyranosy.-(1 leads to 2)-D-glyceric acid was confirmed by chemical and enzymic methods. 13C and 1H NMR spectra of this compound, together with spectra of several disaccharides, were obtained for future reference in the polysaccharide study. The nine resonances in the 13C spectrum were assigned by comparison with the spectrum of methyl alpha-D-glucopyranoside. Analysis of the 1H NMR spectrum showed that the two methylene protons on C-3 of the glycerate moiety were less equivalent in the sodium salt than in the acid. This may be attributable to hydrogen bonding between the carboxylate and the hydrogen atom of the glycerate 3-hydroxyl group.     

Evidence from NMR interaction studies challenges the hypothesis of direct lipid transfer from L‐FABP to malaria sporozoite protein UIS3

UIS3 is a malaria parasite protein essential for liver stage development of Plasmodium species, presumably localized to the membrane of the parasitophorous vacuole formed in infected cells. It has been recently proposed that the soluble domain of UIS3 interacts with the host liver fatty acid binding protein (L‐FABP), providing the parasite with a pathway for importing exogenous lipids required for its rapid growth. This finding may suggest novel strategies for arresting parasite development. In this study, we have investigated the interaction between human L‐FABP and the soluble domain of Plasmodium falciparum UIS3 by NMR spectroscopy. The amino acid residue‐specific analysis of ¹H,¹⁵N‐2D NMR spectra excluded the occurrence of a direct interaction between L‐FABP (in its unbound and oleate‐loaded forms) and Pf‐UIS3. Furthermore, the spectrum of Pf‐UIS3 was unchanged when oleate or phospholipids were added. The present investigation entails a reformulation of the current model of host‐pathogen lipid transfer, possibly redirecting research for early intervention against malaria.     

A 2H NMR study on alteration of the membrane organization of mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate

In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18,18,18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.     

Effect of ConA—receptor interaction on the structure of cell membrane

Recently,the effect of ligand receptor interaction on the membrane structure of liposomes has been studied extensively,However,little is known about how it exists on biological membranes,In this paper,the effect of Concanavalin A(ConA) receptorinteratcion on the structure of cell membranes was studied by Circular DIchrosim(CD) and 31P Nuclear Magnetic Resonance(NMR).CD results of both the purified macrophage membranes and human erythrocyte hgosts(EG) showed that the conformation of membrane proteins changed after ConA binding.For further research,31P-NMR was used to detect the orgainzation of phosp[holipid molecules on macrophage membranes.After ConA binding,the tendercy to form non bilayer structure increased with the amount of ConA.The changes of 31P-NMR spectra of living macrophages might be partly due to the above stated reason too.In addition,ConA-receptor interaction also induced similar results of 31P-NMR spectra in EG.In contrast,wheat germ agglutinin (WGA),another kind of lectin,rarely showed the same influence.     

Structure and dynamics of the phospholipase C-delta1 pleckstrin homology domain located at the lipid bilayer surface

Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the beta5/beta6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic alpha-helix located in the beta5/beta6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-delta1.     

PICK1中N端酸性区域对其PDZ结构域脂质结合能力的调节

蛋白激酶Cα相互作用蛋白1(protein interacting with Cα kinase 1, PICK1)是衔接膜上受体和蛋白激酶Cα的重要蛋白.利用荧光光谱结合定点突变技术 、蛋白与脂质覆盖法等方法,分析了PICK1蛋白N末端区域几个酸性氨基酸残基对PDZ 结构域与膜脂结合的影响,以及钙离子结合N末端酸性区域对PDZ脂结合能力的调节. 结果显示, 带有上游酸性区域的PDZ结构域（NPDZ）的脂质结合能力仅相当PDZ结构 域的15%,相比单独的PDZ结构域与脂质的解离常数Kd(PDZ)为1.58×103 μg·L-1, NPDZ与脂质解离常数Kd(NPDZ)为3.3×104μg·L-1,其中在N末端酸性残基中D8与 D12两个天冬氨酸是影响脂质结合能力减弱的关键残基,若将二者分别突变为丙氨酸 后,NPDZ与脂质的解离常数分别为：Kd (D8/A)=4.42×103μg·L-1;Kd (D12/A) =1.73×103μg·L-1接近于PDZ结构域与脂质结合能力;钙离子会增强NPDZ脂结合能力,当钙离子浓度达到30 μmol/L时,NPDZ的脂结合能力提高2.3倍,但只相当于PDZ的50% 的结合能力.     

Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-delta1

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-delta1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state (13)C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5-bisphosphate (PIP(2)) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic alpha2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP(2) (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the alpha2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP(2) vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl(2), suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.     

Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.     

The conformation of peptide thymosin α1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. a possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

A 2H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate ²H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. ²H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.