Amphipathic antimicrobial piscidin in magnetically aligned lipid bilayers

The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. ³¹P NMR and double-resonance ¹H/¹⁵N NMR experiments performed between 25°C and 61°C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61°C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

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.     

The presence of membranes or micelles induces structural changes of the myristoylated guanylate-cyclase activating protein-2

Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca²⁺ sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.     

Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state ³¹P and ²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically ¹³C-labelled Aβ(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely ¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state ¹H/¹⁵N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.     

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles

GCAPs are neuronal Ca^2 +-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but ²H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca^2 +-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca^2 + and membrane mimetics.     

Solid-State H NMR Shows Equivalence of Dehydration and Osmotic Pressures in Lipid Membrane Deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S_CD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly <Superscript>15</Superscript>N labeled integral membrane proteins in magnetically aligned lipid bilayers

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring ¹H-¹⁵N dipolar couplings (DC) and ¹⁵N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([¹H,¹⁵N]-SE-PISEMA-PDSD). The incorporation of 2D ¹⁵N/¹⁵N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the ¹⁵N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.     

Dynorphin induced magnetic ordering in lipid bilayers as studied by P NMR spectroscopy

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T_m=24°C), as examined by ³¹P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T_m but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T_m. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T_m in parallel with the bilayer surface, because a single ³¹P NMR signal appeared at the perpendicular position of the ³¹P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T_m, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated ³¹P NMR lineshape.     

Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+

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.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

Solid-state NMR and molecular dynamics simulations reveal the oligomeric ion-channels of TM2-GABAA stabilized by intermolecular hydrogen bonding

The second transmembrane (TM2) domain of GABA_A receptor forms the inner-lining surface of chloride ion-channel and plays important roles in the function of the receptor protein. In this study, we report the first structure of TM2 in lipid bilayers determined using solid-state NMR and MD simulations. The interatomic ¹³C-¹⁵N distances measured from REDOR magic angle spinning experiments on multilamellar vesicles, containing a TM2 peptide site specifically labeled with ¹³C′ and ¹⁵N isotopes, were used to determine the secondary structure of the peptide. The ¹⁵N chemical shift and ¹H-¹⁵N dipolar coupling parameters measured from PISEMA experiments on mechanically aligned phospholipid bilayers, containing a TM2 peptide site specifically labeled with ¹⁵N isotopes, under static conditions were used to determine the membrane orientation of the peptide. Our results reveal that the TM2 peptide forms an alpha helical conformation with a tilted transmembrane orientation, which is unstable as a monomer but stable as pentameric oligomers as indicated by MD simulations. Even though the peptide consists of a number of hydrophilic residues, the transmembrane folding of the peptide is stabilized by intermolecular hydrogen bondings between the side chains of Ser and Thr residues as revealed by MD simulations. The results also suggest that peptide-peptide interactions in the tilted transmembrane orientation overcome the hydrophobic mismatch between the peptide and bilayer thickness.     

Structure,topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers

Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch. Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra. On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following amphibian peptides: (1) aurein 1.2, a 13-residue peptide; (2) citropin 1.1, with 16 residues; and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt amphipathic -helical structures in the presence of lipid micelles and vesicles. Specifically ¹⁵N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by ²H and ³¹P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane -helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a "detergent"-like mechanism of membrane lysis.This paper was submitted as a record of the 2002 Australian Biophysical Society     

A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR. ¹³C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala₉ interact with the membrane surface. Order parameters for Cα and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static ²H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from ²H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.