Presence Of SDS Micelles Research Articles

Hydrophobicity and helicity of membrane-interactive peptides containing peptoid residues.

Peptoid (N-alkylglycyl) residues in peptides have been studied in a variety of applications, but their behavior in membrane environments has not been systematically investigated. We have synthesized a series of membrane-interactive peptides of prototypic structure KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X corresponds to the peptoid residues Nala (= sarcosine), Nval, Nile, Nleu, Nphe, and Ntrp. Investigation of their relative hydrophobic character by high-performance liquid chromatography indicated an order of hydrophobicity Ntrp > Nphe > Nleu > Nile > Nval > Nala-largely parallel to the relative scale for these side-chains in natural amino acids, although all values were significantly more "hydrophilic" than their amino acid correspondents. Conformations of peptoid-containing peptides measured by circular dichroism spectroscopy were unordered in the presence of SDS micelles but helical for peptides with X = the corresponding amino acids, suggesting a general helix-breaking tendency for the peptoid residues. However, peptides were able to form helical structures in the solvent n-butanol, indicating that this conformation is possible if peptides became inserted into micellar phases. The latter notion was confirmed by increasing hydrophobic content of the peptides by embedding peptoid Nala residues in Leu-rich rather than Ala-rich sequences, which promoted peptide insertion and helical structure in micelles. The overall results suggest that judicious interspersing of amino acid and peptoid residues in peptide sequences can produce hydrophobic water-soluble materials with membrane-partitioning capacity.

O-Linked glycopeptides retain helicity in water.

A 17-residue O-linked glycopeptide model incorporating a central alpha-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.

NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate.

The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.

The Topology of Lysine-Containing Amphipathic Peptides in Bilayers by Circular Dichroism, Solid-State NMR, and Molecular Modeling

In order to better understand the driving forces that determine the alignment of amphipathic helical polypeptides with respect to the surface of phospholipid bilayers, lysine-containing peptide sequences were designed, prepared by solid-phase chemical synthesis, and reconstituted into membranes. CD spectroscopy indicates that all peptides exhibit a high degree of helicity in the presence of SDS micelles or POPC small unilamellar vesicles. Proton-decoupled 31P-NMR solid-state NMR spectroscopy demonstrates that in the presence of peptides liquid crystalline phosphatidylcholine membranes orient well along glass surfaces. The orientational distribution and dynamics of peptides labeled with 15N at selected sites were investigated by proton-decoupled 15N solid-state NMR spectroscopy. Polypeptides with a single lysine residue adopt a transmembrane orientation, thereby locating this polar amino acid within the core region of the bilayer. In contrast, peptides with ≥3 lysines reside along the surface of the membrane. With 2 lysines in the center of an otherwise hydrophobic amino acid sequence the peptides assume a broad orientational distribution. The energy of lysine discharge, hydrophobic, polar, and all other interactions are estimated to quantitatively describe the polypeptide topologies observed. Furthermore, a molecular modeling algorithm based on the hydrophobicities of atoms in a continuous hydrophilic-hydrophobic-hydrophilic potential describes the experimentally observed peptide topologies well.

Analysis of cyanogenic glycosides by micellar capillary electrophoresis

The separation of amygdalin, prunasin and their isomers neoamygdalin and sambunigrin could be achieved with micellar capillary electrophoresis (MEKC). The two isomers were obtained in alkaline conditions and were produced in less than 15 min at pH 11.0. The developed methods showed a good selectivity in the separation of the isomers only in the presence of SDS micelles. The working pH was optimized to allow best resolution and quantitative analysis of these compounds. With a linear calibration over an injection time from 1 to 20 s, the detection limit was found to be in the range of 5 μM ( S/ N=3; 20 s injection time). Two pH buffer systems (pH 5.2 and pH 9.1) were chosen to confirm the peak attributions of the compounds in the apple and peach seeds samples. Sambunigrin was found in both apple and peach seeds but could not be quantified because of missing standards. Prunasin and amygdalin were not found in the apple sample, while they were quantified in the peach seeds in concentrations of 50 μg/g and 90 μg/g (dry weight), respectively.

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6–11× higher hemolytic activity and 10× higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic α-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3–4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic α-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Gs 1.

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix.

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.

Photoinitiation of MMA polymerization by thionine/amine systems in the presence of SDS micelles

The photoinitiation of the polymerization of methylmethacrylate (MMA) by the dye thionine in the presence of triethylamine and triethanolamine has been studied in the presence of SDS micelles, and compared with similar studies in homogeneous methanol solutions. The conversion rates in the presence of micelles were significantly faster than in the homogeneous solutions, and the amine concentrations at which the maximum rates were observed (∼0.7) were higher than in methanol (∼0.1). Association constants of the dye, monomer, and amines to the micelles were determined, as well as the quenching rate constants of the dye's excited states by the monomer and the amines in these systems. An overall mechanism is proposed for the production of amine free radicals which includes the postulation of a triplex. An expression for the concentration of the initiating radicals was deduced from this mechanism, and when using the experimentally determined constants the fit to the polymerization data is quite good for amine concentrations up to the maximum conversion rate. At higher amine concentrations other processes may be participating in the process decreasing the efficiency of the production of free radicals.

Stable β-phosphorylated cyclic aminoxyl radicals in SDS micelles

The behaviour of a series of stable cyclic aminoxyl radicals in the presence of SDS micelles was studied by EPR spectroscopy. Six different compounds, i.e. two non-phosphorylated and four β-phosphorylated, were investigated. Except in the case of the strongly hydrophilic 3-CP 1, which always remained in the bulk aqueous phase, all the radicals were found to exchange between water and micelles. Their partition coefficients were evaluated from computer simulations of the EPR spectra and in the case of two aminoxyls TOMER-Et 5 and TOMER-Pri 6, the variation of the rate constant with temperature allowed us to estimate the exchange activation energy.

ChemInform Abstract: Spin‐Trapping of Free Radicals by PBN‐Type β‐Phosphorylated Nitrones in the Presence of SDS Micelles

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Spin-trapping of free radicals by PBN-type β-phosphorylated nitrones in the presence of SDS micelles †

Three PBN-type β-phosphorylated nitrones, PPN 1, 4-PyOPN 2, and 4-ClPPN 3, have been used to trap two carbon centred free radicals, CH3˙ and CO2˙–, in a water–SDS biphasic system. The location of the traps and their adducts has been found by EPR spectroscopy. 4-PyOPN is found to remain in water, while 4-ClPPN is preferentially sequestered into the micellar structure, and PPN partitioned between the two phases. With the three nitrones, the CO2˙– spin adduct always remains in the bulk aqueous phase. In contrast, the three CH3˙ spin adducts seem to be present in both phases. However, 4-PyOPN-CH3 is essentially located in water, and 4-ClPPN–CH3 in the micelles, while PPN–CH3 is clearly shown to partition between the aqueous environment and the micellar interior. For all these spin adducts, the phosporus hyperfine splitting constant, aP, is found to be a good indicator of aminoxyl behaviour in the presence of micelles. For the various sodium dodecyl sulfate concentrations, the PPN–CH3 affinity for the micellar phase was evaluated from the average aP value.

Evidence for α-Helical Conformation of an Essential N-terminal Region in the Human Bcl2 Protein

A region occupying approximately 24 amino acids near the N terminus of human Bcl2 is essential for this cytoplasmic membrane protein's ability to inhibit apoptosis. Systematic mutagenesis of this N-terminal region indicates that only five hydrophobic and aromatic residues within it are specifically required for function. Computerized secondary structure prediction, together with circular dichroism spectroscopy of synthetic peptides, indicates that the region encompassing these five residues has the propensity to take on an alpha-helical conformation in the presence of SDS micelles, which presumably mimic the hydrophobic surfaces of cellular membranes or polypeptides. The five critical residues are predicted to be clustered on one face of this putative helix, where they might serve to mediate protein-protein contacts involved in the multimerization of Bcl2 or in the interaction of Bcl2 with other, as yet unidentified components of the apoptotic pathway. Apparent structural homologues of this helical motif are also present in at least some other anti-apoptotic proteins from the Bcl2 family but not in those family members that tend to potentiate, rather than inhibit, apoptosis.

Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy.

Factor VIII binds to cell membranes prior to assembling with the serine protease, factor IXa, to form the factor X-activating enzyme complex. In order to better understand the interaction between factor VIII and phosphatidylserine-containing membranes, we have synthesized the membrane-binding peptide from the C2 domain of factor VIII, corresponding to residues 2303-2324. The peptide, fVIII2303-24, with a primary structure of TRYLRIHPQSWVHQIALRMEVL, aggregates at concentrations above 2 microM at pH 7 but is soluble at pH 6. fVIII2303-24 competes with fluorescein-labeled factor VIII (Ki = 3 microM) for binding sites on synthetic phosphatidylserine-containing membranes and for binding sites on stimulated platelets. Circular dichroism spectra indicate that fVIII2303-24 is predominantly a random coil in aqueous solution but adopts a predominantly helical conformation upon interaction with SDS micelles. 1H NMR spectroscopy in the presence of SDS micelles allowed estimation of interproton distances from the nuclear Overhauser effect and estimation of torsion angles from coupling constants indicated by splitting of resonance lines. The distance and angle estimates, processed by distance geometry/simulated annealing software, indicate that fVIII2303-24 has an alpha-helical segment encompassing residues P8-E20 and an extended segment encompassing residues L4-P8. The location of six hydrophobic residues on one face of the structure suggests that hydrophobic interactions contribute to membrane-binding. In addition, two arginines penetrate the hydrophobic plane suggesting that they interact with phosphate moieties in a phospholipid bilayer.

A dramatic change in the interaction of Cu(II) with bio-peptides promoted by SDS—a model for complex formation on a membrane surface

The extent of complex formation between Cu(II) and many biologically active oligopeptides has been shown to change significantly in the presence of SDS micelles, a recognized model for cell lipid membranes. Protonation constants of peptides can be increased by up to 2 log unit, especially when they contain hydrophobic side chains. Metal complex formation is generally less extensive and the conformations of peptides can be altered dramatically when compared to those in simple aqueous solution.

Protonation of the triplet state of safranine in the presence of cationic micelles and polyelectrolytes

Abstract The protonation of the triplet state of safranine has been studied in the presence of SDS micelles and poly(styrenesulfonate) and poly(vinysulfonate) by laser flash photolysis, measuring the decay kinetics of the absorption of the monoprotonated triplet. The observed second-order rate constant were 2.1×10 11 , 2.9×10 10 and 4.1×10 9 M −1 s −1 respectively. These results indicate that the local concentration of protons, induced by the micelles and the polyelectrolytes, is increased by factors of around 100 for SDS and 10 for PSS. PVS seems not to affect these concentrations.

A peptide corresponding to the N-terminal 13 residues of T4 lysozyme forms an α-helix

Solid-phase methods have been used to synthesize LYS(1–13), a peptide corresponding to the first 13 residues of T4 lysozyme. 2D 1H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium-range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an α-helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.

N-terminal domain of Avena phytochrome: interactions with sodium dodecyl sulfate micelles and N-terminal chain truncated phytochrome.

Phytochrome is the ubiquitous red light photoreceptor present in plants. Properties of the 6-kDa end terminal region of phytochrome A (PHYA from etiolated Avena) have been investigated by the use of synthetic polypeptide fragments corresponding to that region. This region of the phytochrome A protein has been viewed as a possible functional site due to the large differences in the sequence's conformation and exposure between the Pr (red light-absorbing form) and Pfr (far-red light-absorbing, gene-regulating form) species of phytochrome A. Hydrophobic moment calculations reveal amphiphilic helical potential in this section of the protein, consistent with the folding of the N-terminal region onto a hydrophobic chromophore/chromophore pocket. A large N-terminal synthetic peptide also demonstrated helical folding in the presence of SDS micelles. This experimental evidence indicates that the N-terminal alpha-helical folding upon conversion of the regulatorily inactive Pr to the active Pfr form of phytochrome A is likely driven at least in part by amphiphilic helix stabilization. Further, the large synthetic peptide was spectrally demonstrated to interact with phytochrome A lacking the N-terminal region. The formation of this nativelike complex may provide us with a tool for both biophysical and physiological studies on the mechanism of phytochrome A signal transduction.

Conformation of uteroglobin fragments.

The conformation of three fragments of uteroglobin in aqueous solution and in the presence of SDS micelles is described. Two of these fragments correspond to helix II and helix III of uteroglobin, the crystal structure of which is made of four helices. The third peptide comprises helices II and III, with the connecting beta-turn. While helix II does not interact strongly with the micelles, helix III adopts a rather clear alpha-helix in this system. The elongation of helix III with the addition of helix II at the N-terminus somewhat stabilizes the ordered structure. It is possible that the beta-turn found in the crystal is also present in solution.

Conformational studies by circular dichroism, proton NMR, and computer simulations of bombolitins I and III in aqueous solution containing surfactant micelles

The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.