DPC Micelles Research Articles

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-α (LcnG-α; contains 39 residues) and lactococcin G-β (LcnG-β, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-α has an N-terminal α-helix (residues 3–21) that contains a GxxxG helix–helix interaction motif (residues 7–11) and a less well defined C-terminal α-helix (residues 24–34), and in between (residues 18–22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-β has an N-terminal α-helix (residues 6–19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18–22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined α-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-α and -β, are amphiphilic. We postulate that LcnG-α and -β have a parallel orientation and interact through helix–helix interactions involving the first GxxxG (residues 7–11) motif in LcnG-α and the one (residues 18–22) in LcnG-β, and that they thus lie in a staggered fashion relative to each other.

Effect of micelle interface on the binding of anticoccidial PW2 peptide

PW2 is an anticoccidial peptide active against Eimeria acervulina and Eimeria tenella. We determined the structure of PW2 in dodecylphosphocholine micelles. The structure showed two distinct regions: an amphipathic N-terminal 3(10) helix and an aromatic region containing WWR interface-binding motif. The aromatic region acted as a scaffold of the protein in the interface and shared the same structure in both DPC and SDS micelles. N-terminal helix interacted with DPC but not with SDS interface. Chemical shift change was slow when SDS was added to PW2 in DPC and fast when DPC was added to PW2 in SDS, indicating that interaction with DPC micelles was kinetically more stable than with SDS micelles. Also, DPC interface was able to accommodate PW2, but it maintained the conformational arrangement in the aromatic region observed for SDS micelles. This behavior, which is different from that observed for other antimicrobial peptides with WWR motif, may be associated with the absence of PW2 antibacterial activity and its selectivity for Eimeria parasites.

Structural biology of membrane-acting peptides: Conformational plasticity of anticoccidial peptide PW2 probed by solution NMR

The bottleneck for the complete understanding of the structure–function relationship of flexible membrane-acting peptides is its dynamics. At the same time, not only the structure but also the dynamics are the key points for their mechanism of action. Our model is PW2, a TRP-rich, cationic peptide selected from phage display libraries that shows anticoccidial activity against Eimeria acervulina. In this manuscript we used a combination of several NMR techniques to tackle these difficulties. The structural features of the membrane-acting peptide PW2 was studied in several membrane mimetic environments: we compared the structural features of PW2 in SDS and DPC micelles, that were reported earlier, with the structure properties in different lipid vesicles and the peptide free in water. We were able to unify the structural information obtained in each of these systems. The structural constraints of the peptide free in water were fundamental for the understanding of plasticity necessary for the membrane interaction. Our data suggested that the WWR sequence is the region responsible for anchoring the peptide to the interfaces, and that this same region displays some degree of conformational order in solution. For PW2, we found that affinity is related to the aromatic region, by anchoring the peptide to the membrane, and specificity is related to the N- and C-termini, which are able to accommodate in the membrane due to its plasticity.

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air–water interface

Cateslytin (bCGA (344)RSMRLSFRARGYGFR(358)), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a alpha-helical character in the "membrane mimicking" solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A beta-sheet contribution of almost 100% is detected at the air-water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin.

Recombinant expression, synthesis, purification, and solution structure of arenicin

Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged β-hairpin, formed by two antiparallel β-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the β-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.

19F NMR detection of the complex between amantadine and the receptor portion of the influenza A M2 ion channel in DPC micelles

19F NMR probes were used to follow interactions between ligands in the aminoadamantane series, amantadine (Am) 1 and 3-F-Am 2, and the 5-F-Trp20 transmembrane fragment of the influenza A M2 proton channel (F-M2TM 3) in dodecylphosphocholine micelles over the pH range 5–8. Above pH 7, when the peptide adopts a tetrameric state that is able to bind channel blocking ligands, 19F-Trp signals from both the free and bound states of the M2TM tetramer are resolved. This differentiation of bound and unbound states of the M2TM receptor by 19F NMR may provide a system for SAR studies.

Investigation of the micellar growth of 1-dodecylpyridinium chloride in aqueous solution of phenol

Viscosity and conductivity measurements have been made, in a series of concentrations and temperatures, to study the effect of phenol on the micellar growth of 1-dodecylpyridinium chloride (DPC) up to very high surfactant concentration. From the data the first and second critical micelle concentrations, cmc 1 and cmc 2, and the degree of counter-ion binding, β, of the micelles were determined. The focus has been on finding the effect of high surfactant concentration and various additive concentrations on the growth of DPC micelles at different temperatures which has been correlated with the size and shape of micelles formed. The results indicate that (i) the shape transition of micelles from sphere-to-rod occurs at high DPC concentration, (ii) both micelle formation and micellar growth are accelerated by phenol due to its solubilization into the micelles, and (iii) temperature plays an important role in de-micellization, thus increasing cmc 1 and cmc 2, in addition to impeding micellar growth.

Copper binding and conformation of the N‐terminal octarepeats of the prion protein in the presence of DPC micelles as membrane mimetic

The prion protein is usually pictured as globular structured C-terminal domain that is linked to an extended flexible N-terminal tail. However, in its physiological form, it is a glycoprotein tethered to the cell surface via a C-terminal GPI anchor. The low solubility of PrP even without GPI anchor and its strong tendency for aggregation has forced most structural investigations to be performed at low pH and mostly with N-terminally truncated variants. In the present study, we have used a synthetic peptide related to the PrP tetra-octarepeat region, i.e., the sequence (Pro-His-Gly-Gly-Gly-Trp-Gly-Gln)(4), for NMR structural analysis of its preferred conformation in DPC micelles as membrane mimic. Well-defined and identical loops are observed between the four octarepeats that are linked by flexible Gly-Gly-Gly sequences. Interaction with the micelles is mainly through the tryptophan residues that appear to act as anchors. Copper binding to the peptide in the presence of DPC micelles revealed marked conformational rearrangements although binding to the micelles is preserved. Interestingly, titration experiments point to cooperative effects for the four binding sites. A destabilization of the DPC micelles by the peptide parallels the destabilizing effect of the prion protein on membranes so that the octarepeat region appears to be very membrane-active. How the physico-chemical properties reported here are linked to the function and significance of the prion protein remains a puzzle as long as the functional mechanism of the prion protein is not precisely elucidated. Nevertheless, our results emphasize the strong influence of the (membrane) environment on the PrP properties.

Solution structure of a novel D‐naphthylalanine substituted peptide with potential antibacterial and antifungal activities

A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH2, designated as Pac-525, was found to possess improved activity against both gram-positive and negative bacteria. We have synthesized two Pac-525 analogues, D-Pac-525 containing all D-amino acids and D-Nal-Pac-525, the D-Pac-525 analogue with tryptophan replaced by D-beta-naphthylalanine. We have determined the solution structure of D-Nal-Pac-525 bound to membrane-mimetic DPC micelles by two-dimensional NMR methods. The DPC micelle-bound structure of D-Nal-Pac-525 adopts a left-hand alpha-helical segment and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates the three D-beta-naphthylalanines are packed against the peptide backbone and form an amphipathic structure. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that D-Nal-Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the strong antimicrobial activity of D-Nal-Pac-525 may be due to interactions with bacterial and fungus membranes.

Membrane-Bound Conformation and Topology of the Antimicrobial Peptide Tachyplesin I by Solid-State NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.

Conformationally Constrained CCK8 Analogues Obtained from a Rationally Designed Peptide Library as Ligands for Cholecystokinin Type B Receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems

P59 is the Trp-rich 20-mer peptide ( 767L–G 786), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.

Membrane-Bound Dimer Structure of a β-Hairpin Antimicrobial Peptide from Rotational-Echo Double-Resonance Solid-State NMR

The intermolecular packing of a beta-hairpin antimicrobial peptide, PG-1, in lipid bilayers is determined using solid-state NMR distance measurements. Previous spin counting experiments showed that PG-1 associates as dimers in POPC bilayers; however, the detailed dimer structure was unknown. We have now measured several intermolecular 13C-19F, 1H-13C, and 15N-13C distances in site-specifically labeled PG-1 to constrain the structure of the intermolecular interface. The distances are measured using the rotational-echo double-resonance (REDOR) technique under magic-angle spinning. The results indicate that two PG-1 molecules align in a parallel fashion with the C-terminal strand of the hairpin forming the dimer interface. Six hydrogen bonds stabilize this interface, and the Phe12 side chain adopts the g- conformation in the membrane as in solution. The parallel packing of the peptide in the lipid bilayer differs from the antiparallel dimer found in DPC micelles and may be stabilized by its strong amphipathic character, which should facilitate its insertion into the amphipathic lipid bilayer. This study demonstrates the utility of the REDOR NMR technique for the elucidation of the oligomeric structure of membrane proteins.

A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

The cannabinoid receptor subtype 2 (CB2) is a member of the G-protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double-helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC-purified protein in solution and in DPC micelle preparations suggested predominant alpha-helical structures under both conditions. The 13C/15N double-labeled protein CB2(27-101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The 15N HSQC NMR results show a clear chemical shift dispersion of the amide nitrogen-proton correlation indicative of a pure double-labeled polypeptide molecule. The results suggest that this method is capable of generating transmembrane helical bundles from GPCRs in quantity and purity sufficient for NMR and other biophysical studies. Therefore, the biosynthesis of GPCR transmembrane helix bundles represents a satisfactory alternative strategy to obtain and assemble NMR structures from recombinant "building blocks."

Invariant Chain Transmembrane Domain Trimerization: A Step in MHC Class II Assembly

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.

Effects of mutations on the C-terminus of protegrin-1: a molecular dynamics simulation study

In this work the effects of the charge of the C-terminus of protegrin-like peptides on activity and toxicity are examined by molecular dynamics simulations. Simulations are done in sodium dodecylsulphate and dodecylphosphocholine micelles, bacterial and mammalian membrane mimics, respectively. Three protegrin mutants are examined and it is found that while the peptides interact in different ways, the peptides all insert into the SDS micelles equally as deep, in agreement with their equal activities as determined by previous experimental work. There are clear differences in the interactions with the DPC micelles and it is demonstrated that simulations with DPC micelles can predict levels of toxicity of such peptides. We also see that removing the positive charge from the sequence of protegrin-1 does not have positive effects on the resulting peptide's toxicity, but that replacing the positive charge with a negative charge reduces the toxicity.

Conformation and Environment of Channel-Forming Peptides: A Simulation Study

Ion channel-forming peptides enable us to study the conformational dynamics of a transmembrane helix as a function of sequence and environment. Molecular dynamics simulations are used to study the conformation and dynamics of three 22-residue peptides derived from the second transmembrane domain of the glycine receptor (NK 4-M2GlyR-p22). Simulations are performed on the peptide in four different environments: trifluoroethanol/water; SDS micelles; DPC micelles; and a DMPC bilayer. A hierarchy of α-helix stabilization between the different environments is observed such that TFE/water < micelles < bilayers. Local clustering of trifluoroethanol molecules around the peptide appears to help stabilize an α-helical conformation. Single (S22W) and double (S22W,T19R) substitutions at the C-terminus of NK 4-M2GlyR-p22 help to stabilize a helical conformation in the micelle and bilayer environments. This correlates with the ability of the W22 and R19 side chains to form H-bonds with the headgroups of lipid or detergent molecules. This study provides a first atomic resolution comparison of the structure and dynamics of NK 4-M2GlyR-p22 peptides in membrane and membrane-mimetic environments, paralleling NMR and functional studies of these peptides.

High reolution structural studies on peripherin, an intergal membrane protein

Peripherin is a tetraspannin transmembrane protein localized in retinal rod outer segment disk membranes whose complete 3-dimensional structure is the subject of this nuclear magnetic resonance (NMR) analysis. Peripherin promotes membrane fusion and may be involved in fusion events with rod cell membranes during the rod outer segment disk renewal processes. The 63-residue C-terminal extramembraneous region of peripherin (PERCTER) is crucial to peripherin-mediated fusion and contains both an incisure affinity region and an OS targeting signal. Several mutations in this C-terminal region have been linked to various forms of retinal degenerative disease. The first target of our structural analysis has been the PERCTER, expressed and labeled with 15N, and subjected to structural determination by solution NMR. For this analysis, 3-dimensional HSQC-NOESY and HSQC-TOCSY experiments were utilized, along with 2-dimensional 1H NOESY and TOCSY experiments at various mixing times. This analysis is followed by a similar study of the PERCTER in the presence of a membrane-mimetic, DPC micelles. The next stage of our examination is to obtain NMR data from the isotope-labeled full length peripherin. Detailed understanding of the peripherin structure provides greater insight into retinal degenerative diseases and the mechanisms of membrane fusion. NIH EY10420-14, E. Matilda Ziegler Vision Award.

An NMR‐Based Binding Assay Used To Screen For Compounds That Bind To Cytochrome P450 Enzymes

The cytochromes P450 are the enzymes predominantly responsible for the metabolism of drugs and other exogenous compounds that enter into our bodies. Because of these enzymes’ prominent role, it would be helpful to be able to quickly predict which compounds—potential drug candidates, toxins, or protoxins—bind to these enzymes, thus providing invaluable data to serve as the starting point for later, more thorough studies. This quick prediction can be accomplished in the form of a high-throughput assay that involves monitoring the 13C-attached 1H chemical shift (13C-attached 1H δ) and 13C δ of 13CH3NC (methyl isocyanide). As is the case for any short-chain alkylisocyanide, methyl isocyanide binds through its isonitrile moiety to the heme iron contained within the active site of the cytochrome P450 enzymes. We show here, in a series of 13C-1H HSQC spectra and for several cytochrome P450s, that the two previously mentioned chemical shifts of this ligand change in going from the free (unbound) form to the bound form and then, again upon addition of a known substrate (distinct from both bound and unbound). In addition, for the two full-length, membrane-bound P450s, we show that the chemical shifts indicative of the bound form change depending on whether the protein has been solubilized in 20% (v/v) glycerol or incorporated into DPC micelles.

Three-Dimensional Structure of Neuropeptide K Bound to Dodecylphosphocholine Micelles

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.