Presence Of Dodecylphosphocholine Research Articles

A revisit of the conformational dynamics of SNARE protein rYkt6

N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. R-SNARE protein Ykt6 is one of the most conserved SNARE in eukaryotes. The conformational state of Ykt6 is regulated by the lipidations at its C-terminal motif. Previous studies show that the binding of dodecylphosphocholine (DPC) can stabilize a closed conformation of rat Ykt6 (rYkt6) and mimic the farnesylated rYkt6. Despite this model, the detailed conformational dynamics of Ykt6 is still unclear. Here, we combined smFRET and MD simulation to demonstrate that the un-lipidated rYkt6 adopts five major conformational states. DPC binding shifts the conformational distribution toward the more closed states. At the same time, there remain considerable fractions of open and semi-open conformations in the presence of DPC. These newly revealed dynamic features of rYkt6 are consistent with its unique functional diversity in neuronal cells.

Structural Basis of Membrane Targeting by the Innate Immunity Adaptor TIRAP

Toll-like receptors (TLRs) are responsible for early immune system recognition and response to infection. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade though adaptor proteins, starting with the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal phosphoinositide (PIP)-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane to activate the TLR signaling pathway. To understand the mechanism of TIRAP membrane targeting and the basis for its regulation, we functionally and structurally characterized TIRAP and its PBM using biophysical approaches. We show that TIRAP's PBM adopts a helical conformation in the presence of dodecylphosphocholine (DPC) micelles. NMR studies revealed that TIRAP PBM binds PIPs through its two conserved basic termini following a fast-exchange regime with moderate affinity. Mutation of these two basic regions abolishes PIP binding without distorting the helical structure of the peptide. Interestingly, association of TIRAP PBM to PIPs requires the acyl chains of the lipid. The solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas the N- and C- termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that this N- and C-termini of TIRAP PBM become more ordered when bound to PIPs. Thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction.

Membrane Interaction of the Glycosyltransferase WaaG

The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely α-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

Myelin basic protein is a glial microtubule-associated protein – Characterization of binding domains, kinetics of polymerization, and regulation by phosphorylation and a lipidic environment

The 18.5-kDa splice isoform of myelin basic protein (MBP) predominates in the adult brain, adhering the cytoplasmic leaflets of the oligodendrocyte membrane together, but also assembling the cytoskeleton at leading edges of membrane processes. Here, we characterized MBP's role as a microtubule-assembly protein (MAP). Using light scattering and sedimentation assays we found that pseudo-phosphorylation of Ser54 (murine 18.5-kDa sequence) significantly enhanced the rate but not the final degree of polymerization. This residue lies within a short KPGSG motif identical to one in tau, a ubiquitous MAP important in neuronal microtubule assembly. Using polypeptide constructs, each comprising one of three major amphipathic α-helical molecular recognition fragments of 18.5-kDa MBP, we identified the N-terminal α1-peptide as sufficient to cause microtubule polymerization, the rate of which was significantly enhanced in the presence of dodecylphosphocholine (DPC) micelles to mimic a lipidic environment.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1 and its Effects on the Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance (NMR)/molecular dynamics (MD) characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate (SDS) micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups, but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 µg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12 IL-23 and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, while the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1, and Its Effects on Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 μg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Structural Characterization of Hsp12, the Heat Shock Protein from Saccharomyces cerevisiae, in Aqueous Solution Where It Is Intrinsically Disordered and in Detergent Micelles Where It Is Locally α-Helical

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R(1) and R(2), relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.

Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: The stabilizing effect of membrane‐mimetics on the acquired fold

A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an amino-terminal signal peptide. In Escherichia coli, typically signal peptides that guide preproteins into the SecYEG protein conduction channel are subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution nuclear magnetic resonance. We employed a soluble form of signal peptidase, which laks the two transmembrane domains (SPase I Δ2-75), and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of a weak peptide-enzyme complex formation. The peptide adopts a U-turn shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable with that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and based on PRE (paramagnetic relaxation enhancement) experiments are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.

Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G⁎ apolipoprotein J peptide using solution NMR

The surprising observation that a 10-residue class G⁎ peptide from apolipoprotein J, [113–122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113–122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (−16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (−3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (−13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113–122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.

Structural Disorder in the HIV-1 Vif Protein and Interaction-Dependent Gain of Structure

The HIV-1 Vif protein (192 residues) is required for HIV-1 infection of many target cells. Vif overcomes the anti-viral cellular defense by antagonizing the cellular cytosine deaminase APOBEC-3G through impairing APOBEC-3G production, inhibiting its enzymatic activity and targeting it for degradation. Vif interacts with several viral and cellular molecules, particularly via its C-terminal domain (residues 100-192). The structure of full-length Vif has not yet been determined. The structure of Vif and its domains was studied using computational and experimental methods. Computational predictions resulted in two suggested homology models for the full length protein. Experimental studies have shown that the Vif C-terminal domain is mainly unstructured. Residues 108-139 have mainly random coil conformation in the unbound state. This region includes an HCCH Zn(2+)-binding motif that also mediates Vif binding to Cul5, a protein in the E3 ubiquitin ligase complex. The C-terminal domain residues 141-192, which mediate interactions with both ElonginC and Cul5, are intrinsically disordered. This region also includes several phosphorylation sites and regions associated with the ability of Vif to undergo self-oligomerization. The unstructured nature of these regions enables them to interact with several ligands, and probably adopt various conformations as is typical for intrinsically disordered proteins. This was demonstrated by a conformational change induced by Zn(2+) binding to the HCCH motif and a conformational change that the C-terminal domain underwent in the presence of dodecylphosphocholine. The only available crystal structure of Vif includes residues 140-155, which are helical when bound to the ElonginBC complex. Overall, empirical structures, predictions and other experimental data for Vif did not always indicate the same degree or type of structure for any given region. This ambiguity is likely to be the tenet of structurally unfolded proteins, which have the propensity to adopt a multitude of biologically relevant and active conformations.

Interaction of Myelin Basic Protein with Actin in the Presence of Dodecylphosphocholine Micelles

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the myelin sheath in the central nervous system. Protein deimination and phosphorylation are two key posttranslational modifications whose balance determines local myelin microdomain stability and function. It has previously been shown that MBP in solution causes both polymerization of G-actin to F-actin and bundling of the microfilaments, and binds them to a negatively charged membrane. However, the binding parameters, and the roles of different possible interacting domains of membrane-associated MBP, have not yet been investigated. Here, we compared the interaction of unmodified (rmC1) and pseudodeiminated (rmC8) recombinant murine MBP (full-length charge variants), and of two terminal deletion variants (rmDeltaC and rmDeltaN), with actin in the presence of DPC (dodecylphosphocholine) to mimic a membrane environment. Our results show that although both charge variants polymerized and bundled actin, the maximal polymerization/bundling due to rmC1 occurred at a lower molar ratio compared to rmC8. In the presence of DPC, rmC1 appeared to be more active than rmC8 in its ability to polymerize and bundle actin, and the binding affinity of both charge variants to G-actin became higher. Moreover, of the two deletion variants studied in the presence of DPC, the one lacking the C-terminal domain (rmDeltaC) was more active compared to the variant lacking the N-terminal domain (rmDeltaN) but exhibited weaker binding to actin. Thus, whereas the N-terminal domain of MBP can be more important for the MBP's actin polymerization activity and membrane-association, the C-terminal domain can regulate its interaction with actin.

New Insights into the Interfacial Activation of Secreted Phospholipase A2

Despite numerous studies towards elucidation of the structural basis of activation of secreted PLA2s upon membrane binding (interfacial activation), no consistent or clear picture has emerged thus far. Previously we have reported significant changes in the secondary and dynamic structures of human group IB and IIA PLA2s, as well as changes in their mode of membrane binding during activation. Here we have conducted atomic resolution NMR studies on free and phospholipid micelle-bound human group IIA PLA2 (hIIAPLA2) to detect more detailed molecular events underlying interfacial activation. Two-dimensional 1H,15N-HSQC spectra have been obtained at 600 MHz on Ca2+-free and Ca2+-loaded hIIAPLA2 in the presence of dodecylphosphocholine (DPC) micelles. Upon complex formation with the micelles, signals from arginine side chain -NH2 groups of Ca2+-loaded hIIAPLA2 are observed, whereas for Ca2+-free PLA2 these signals are absent because of fast H/D exchange with the solvent. This suggests that the Ca2+-loaded hIIAPLA2 tightly binds to the micelles so these groups are sequestered at the PLA2-micellar interface and shielded from the solvent, or that they are otherwise stabilized by strong hydrogen bonding in the micelle-bound state. TROSY experiments (900 MHz) on Ca2+-loaded, 15N,13C-labeled hIIAPLA2 in the absence and presence of DPC micelles (1:600 protein-to-DPC molar ratio) identify substantial conformational changes in PLA2 upon binding to the micelles. Based on the assigned chemical shifts, important structural changes occur throughout the protein. The molecular mechanism of the strong increase in activity of hIIAPLA2 upon phospholipid surface binding is likely to involve a widening of the substrate binding pocket, mediated by a rigid-body movement of the N-terminal helix via interactions of the cationic residues (e.g., Arg7) with lipid phosphate groups. This mechanism will be tested in further studies and may be shared by other secreted PLA2 isoforms.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 15N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36–Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs–ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Short neuroprotective peptides, ADNF9 and NAP, are structurally disordered and monomeric in PBS

Activity-dependent neurotrophic factor 9 (ADNF9) and NAP are nine and eight amino acid peptides, which exhibit neuroprotective activity at femtomolar concentrations against cell toxic agents. We have here characterized their structures and interactions with dodecylphosphocholine (DPC) in phosphate-buffered saline (PBS). Circular dichroism analysis showed that ADNF9 and NAP are structurally disordered in PBS independent of peptide concentration and temperature, but appear to assume different secondary structure at increasing temperature. Sedimentation equilibrium analysis showed that both ADNF9 and NAP are monomeric at 37 °C, suggesting no self-association under physiological conditions. No secondary structure changes were observed in the presence of DPC, suggesting that ADNF9 and NAP do not interact with lipids.

Studies of the Structure of the N‐Terminal Domain from the Y4 Receptor—a G Protein‐Coupled Receptor—and its Interaction with Hormones from the NPY Family

Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 microM affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor, whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP, and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket.

Role of the membrane interface on the conformation of the caveolin scaffolding domain: A CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82–R101 and D82–I109, encompassing the caveolin scaffolding domain (D82–R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102–I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82–L109 peptide, mainly located in the V 94TKYWFYR 101 motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.

Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy

Structural changes for a series of antimicrobial peptides in various solvents were investigated by a combined approach of FTIR and CD spectroscopy. The well-characterized and potent antimicrobial peptides indolicidin and tritrpticin were studied along with several analogs of tritrpticin, including Tritrp1 (amidated analog of tritrpticin), Tritrp2 (analog of Tritrp1 with Arg → Lys substitutions), Tritrp3 (analog of Tritrp1 with Pro → Ala substitutions) and Tritrp4 (analog of Tritrp1 with Trp → Tyr substitutions). All peptides were studied in aqueous buffer, ethanol and in the presence of dodecylphosphocholine (DPC) micelles. It was shown that tritrpticin and its analogs preferentially adopt turn structures in all solvents studied. The turn structures formed by the tritrpticin analogs bound to DPC micelles are more compact and more conformationally restricted compared to indolicidin. While several peptides showed a slight propensity for an α-helical conformation in ethanol, this trend was only strong for Tritrp3, which also adopted a largely α-helical structure with DPC micelles. Tritrp3 also demonstrated along with Tritrp1 the highest ability to interact with DPC micelles, while Tritrp2 and Tritrp4 showed the weakest interaction.

Consequences of N-Acylation on Structure and Membrane Binding Properties of Dermaseptin Derivative K4-S4-(1-13)

Acyl conjugation to antimicrobial peptides is known to enhance antimicrobial properties. Here, we investigated the consequences of aminolauryl (NC(12)) conjugation to the dermaseptin derivative K(4)-S4-(1-13) (P) on binding properties to bilayer models mimicking bacterial plasma membrane, which is often cited as the ultimate site of action. Isothermal titration calorimetry revealed that acylation was responsible for enhancing the binding affinity of NC(12)-P compared with P (K = 13 x 10(5) and 1.5 x 10(5) m(-1), respectively). Surface plasmon resonance measurements confirmed the isothermal titration calorimetry results (K(app) = 12.6 x 10(5) and 1.53 x 10(5) m(-1), respectively) and further indicated that enhanced adhesion affinity (K(adhesion) = 3 x 10(5) and 1 x 10(5) m(-1), respectively) was coupled to enhanced tendency to insert within the bilayer (K(insertion) = 4.5 and 1.5, respectively). To gain insight into the molecular basis for these observations, we investigated the three-dimensional structures in the presence of dodecylphosphocholine using NMR. The ensemble of NMR-calculated structures (backbone root mean square deviation <0.6 A) showed that the acyl moiety was responsible for a significant molecular reorganization, possibly affecting the electrostatic potential distribution in NC(12)-P relative to that of P. The combined data present compelling evidence in support of the hypothesis that N-acylation affects antimicrobial properties by modifying the secondary structure of the peptide in a manner that facilitates contact with the membrane and consequently increases its disruption.

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes,

The horseshoe crab cationic antimicrobial peptide polyphemusin I is highly active in vitro but not protective in mouse models of bacterial and LPS challenge, while a synthetic polyphemusin variant, PV5, was previously shown to be protective in vivo. In this study, we investigated the interaction of these peptides with lipid membranes in an effort to propose a mechanism of interaction. The solution structure of PV5 was determined by proton NMR in the absence and presence of dodecylphosphocholine (DPC) micelles. Like polyphemusin I, PV5 is a beta-hairpin but appeared less amphipathic in solution. Upon association with DPC micelles, PV5 underwent side chain rearrangements which resulted in an increased amphipathic conformation. Using fluorescence spectroscopy, both peptides were found to have limited affinity for neutral vesicles composed of phosphatidylcholine (PC). Incorporation of 25 mol % cholesterol or phosphatidylethanolamine into PC vesicles produced little change in the partitioning of either peptide. Incorporation of 25 mol % phosphatidylglycerol (PG) into PC vesicles, a simple prokaryotic model, resulted in a large increase in the affinity for both peptides, but the partition coefficient for PV5 was almost twice that of polyphemusin I. Differential scanning calorimetry studies supported the partitioning data and demonstrated that neither peptide interacted readily with neutral PC vesicles. Both peptides showed affinity for negatively charged membranes incorporating PG. The affinity of PV5 was much greater as the pretransition peak was absent at low peptide to lipid ratios (1:400) and the reduction in enthalpy of the main transition was greater than that produced by polyphemusin I. Both peptides decreased the lamellar to inverted hexagonal phase transition temperature of PE indicating the induction of negative curvature strain. These results, combined with previous findings that polyphemusin I promotes lipid flip-flop but does not induce significant vesicle leakage, ruled out the torroidal pore and carpet mechanisms of antimicrobial action for these polyphemusins.

Backbone dynamics of human parathyroid hormone (1–34): Flexibility of the central region under different environmental conditions

The presence of a stable tertiary structure in the bioactive N-terminal portion of parathyroid hormone (PTH), a major hormone in the maintenance of extracellular calcium homeostasis, is still debated. In this work, 15N relaxation parameters of the 33 backbone amides of human PTH(1-34) were determined in phosphate-buffered saline solution (PBS) and in the presence of dodecylphosphocholine (DPC) micelles. The relaxation parameters were analyzed using both the model-free formalism (G. Lipari and A. Szabo, Journal of the American Chemical Society, 1982, Vol. 104, pp. 4546-4549) and the reduced spectral density functions approach (J.-F. Lefevre, K. T. Dayie, J. W. Peng, and G. Wagner, Biochemistry, 1996, Vol. 35, pp. 2674-2686). In PBS, the region around Gly12 possesses a high degree of flexibility and the C-terminal helix is less flexible than the N-terminal one. In the presence of DPC micelles, the mobility of the entire molecule is reduced, but the stability of the N-terminal helix increases relative to the C-terminal one. A point of relatively higher mobility at residue Gly12 is still present and a new site of local mobility at residues 16-17 is generated. These results justify the lack of experimental nuclear Overhauser effect (NOE) restraints with lack of tertiary structure and support the hypothesis that, in the absence of the receptor, the relative spatial orientation of the two N- and C-terminal helices is undefined. The flexibility in the midregion of PTH(1-34), maintained in the presence of the membrane-mimetic environment, may enable the correct relative disposition of the two helices, favoring a productive interaction with the receptor.