High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

A metadynamic approach to understand the recognition mechanism of the histone H3 tail with the ATRXADD domain

The binding affinity between the histone 3 (H3) tail and the ADD domain of ATRX (ATRX_ADD) increases with the subsequent addition of methyl groups on lysine 9 on H3. To improve our understanding of how the difference in methylation state affects binding between H3 and the ATRX_ADD, we adopted a metadynamic approach to explore the recognition mechanism between the two proteins and identify the key intermolecular interactions that mediate this protein-peptide interaction (PPI). The non-methylated H3 peptide is recognized only by the PHD finger of ATRX_ADD while mono-, di-, and trimethylated H3 is recognized by both the PHD and GATA-like zinc finger of the domain. Furthermore, water molecules play an important role in orienting the lysine 9 anchor towards the GATA-like zinc finger, which results in stabilizing the lysine 9 binding pocket on ATRX_ADD. We compared our computational results against experimentally determined NMR and X-ray structures by demonstrating the RMSD, order parameter S² and hydration number of the complex. The metadynamics data provide new insight into roles of water-bridges and the mechanisms through which K9 hydration stabilizes the H3K9me3:ATRX_ADD PPI, providing context for the high affinity demonstrated between this protein and peptide.     

Conformational isomerism of the binuclear N,N-pentamethylenedithiocarbamate cadmium(II) complex, [Cd2{S2CN(CH2)5}4] on multinuclear (N, Cd) CP/MAS NMR and single-crystal X-ray diffraction data

Crystalline N,N-cyclo-pentamethylenedithiocarbamate (PmDtc) cadmium(II) complex was prepared and studied by means of ¹⁵N, ¹¹³Cd CP/MAS NMR spectroscopy and single-crystal X-ray diffraction. The unit cell of the cadmium(II) compound comprises two centrosymmetric isomeric binuclear molecules [Cd₂{S₂CN(CH₂)₅}₄], which display structural inequivalence in both ¹⁵N and ¹¹³Cd NMR and XRD data. There are pairs of the dithiocarbamate ligands exhibiting different structural functions in both isomeric molecules. Each of the terminal ligands is bidentately coordinated to the cadmium atom and forms a planar four-membered chelate ring [CdS₂C]; whereas pairs of the tridentate bridging ligands combine two neighbouring cadmium atoms forming an extended eight-membered tricyclic moieties [Cd₂S₄C₂], whose geometry can be approximated by a ‘chair’ conformation. The structural states of cadmium atoms were characterised by almost axially symmetric ¹¹³Cd chemical shift tensors. All experimental ¹⁵N resonance lines were assigned to the nitrogen structural sites in both isomeric binuclear molecules.     

Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein

A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in −18 Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp–Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H₂¹⁸O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without ¹⁸O incorporation. The light chain peptide L2 VTITCITSTDID₁₂DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp–Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD₂₄DFK. No other isomerizations were detected in CDR peptides containing either Asp–Ser or Asp–Thr motifs.     

Enhancing the production of an antifungal compound from Anabaena laxa through modulation of environmental conditions and its characterization

An investigation was undertaken to optimize the physiological conditions and characterize the bioactive compound responsible for fungicidal activity in Anabaena laxa. Fungicidal activity against Pythium debaryanum in the A. laxa cultures increased, when grown under continuous light (CL) at the stationary phase (28 d), with further enhancement on doubling phosphorus levels and high pH (9.0) in the growth medium. Preparatory thin layer chromatography analyses revealed the peptide nature of the fungicidal metabolite, with highest activity in a spot with R_f 0.13. The bioactive fraction (in terms of fungicidal activity) was isolated and identified using HPLC with a retention time of 14.7. Fourier transform infrared (FT-IR) spectrum of the purified bioactive fraction indicated the cyclic peptide nature of the antifungal compound. The structural elucidation using ¹H, and ¹³C NMR analyses revealed the same number and type of carbons as present in previously reported majusculamide C from Lyngbya majuscula. GC–MS indicated a similar major mass ion fragment spectra, with peaks as previously obtained from majusculamide C. This represents a first report on biosynthesis of a fungicidal compound in A. laxa, with structural similarities to majusculamide C.     

Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [¹³C₁]propionate and [¹³C₃]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[²H₂]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.     

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Two peptides, RAWVAWR-NH₂ and IVSDGNGMNAWVAWR-NH₂, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D ¹H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH₂ peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by ¹H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH₂ interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO₂ fixation and in vivo NH₄⁺ assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO₂ fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.     

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH₂ (HSP1-NH₂), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH₂ with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH₂ presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. ²H exchange experiments of HSP1-NH₂ indicated the insertion of the most N-terminal residues (1–3) in the DPC-d₃₈ micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH₂ (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH₂ peptide depends on the membrane composition and peptide concentration.     

Conformation of adenosine 3′,5′-monophosphate in solution as studied by the NMR-desert method II. Self-association and temperature-dependent glycosidic isomerization at pH 7

A study has been made of the association and the temperature-dependent conformation of adenosine 3′,5′-monophosphate (cyclic AMP) in a neutral aqueous (²H₂O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1′), H(2), and H(8), have enabled us to estimate the self-association constant, K_a = 1.1 ± 0.3 M^?1 at 25°C and thermodynamic parameters ΔH = ?5.8 ± 1.5 kcal/mol and ΔS (25°C) = ?19.0 ± 3 cal/mol per degree.The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity of syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a ‘trans-stacking’ conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.     

Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R² = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase_134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase_134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R² = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase_134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase_134-143 peptide.     

Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by 1H NMR

Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several ¹H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2–9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the ¹H NMR spectra of TNM-A with sodium dodecyl sulfate-d₂₅ (SDS-d₂₅) micelles and small dimyristoylphosphatidylcholine (DMPC)-d₅₄/dihexanoylphosphatidylcholine (DHPC)-d₂₂ bicelles in the presence of a paramagnetic quencher Mn²⁺. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader ¹H signals of TNM-A were observed in 10 mol % cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn²⁺ to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid–water interface (close to the C2′ portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.     

Influence of trifluoroethanol on membrane interfacial anchoring interactions of transmembrane alpha-helical peptides

Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW₂(LA)₈LW₂A-NH₂) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using ²H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3° to 2.5°, as measured by ²H NMR on Ala-d₄ labeled peptides. The “straightening” of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK₂(LA)₈LK₂A-NH₂) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface.     

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C¹X₃C²X_nC³X₃C⁴), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C¹–C⁴ and C²–C³. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H⁺-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY ¹H-¹H NMR spectra are obtained of sMTM7 dissolved in d₆-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two α-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by d-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-^dH containing d-amino acids [^dV5, ^dV8, ^dL11 and ^dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-^dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-^dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-^dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D₂O demonstrated a large difference in dynamics between Mel-H and Mel-^dH, whereby almost all backbone protons of Mel-^dH showed a much faster rate of exchange as compared to Mel-H. Proton T₁ relaxation had suggested a mobile backbone of Mel-^dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-^dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.     

Human neuromodulator SLURP-1: Bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution

Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E. coli. The bacterial systems engineered for SLURP-1 expression as fused with thioredoxin and secretion with leader peptide STII failed in the production of milligram quantities of the protein. The SLURP-1 was produced with high-yield in the form of inclusion bodies, and different methods of the protein refolding were tested. Milligram quantities of recombinant SLURP-1 and its ¹⁵N-labeled analog were obtained. The recombinant SLURP-1 competed with ¹²⁵I-α-bungarotoxin for binding to muscle-type Torpedo californica nAChR at micromolar concentrations, indicating a partial overlap in the binding sites for SLURP-1 and α-neurotoxins on the receptor surface. NMR study revealed conformational heterogeneity of SLURP-1 in aqueous solution, which was associated with cis-trans isomerization of the Tyr39-Pro40 peptide bond. The two structural forms of the protein have almost equal population in aqueous solution, and exchange process between them takes place with characteristic time of about 4 ms. Almost complete ¹H and ¹⁵N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel β-sheets formed from five β-strands and closely resembles those of three-finger snake neurotoxins.