Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.     

Vibrational spectral signatures of peptide secondary structures: N-methylation and side chain hydrogen bond in cyclosporin A

Molecular dynamics simulations are performed to explore important conformations of cyclosporin A, an immunosuppressive cyclic undecapeptide drug, in different media including gas-phase, chloroform, and acetonitrile. Density functional theory calculations are used to refine the low-lying conformers and to predict their infrared and vibrational circular dichroism spectra. Vibrational spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including β-turn (type II' or I), antiparallel β-sheet (flat or twisted), inverse γ-turn, N-methylated peptide bond, and side chain H-bond. New insights into the spectral signatures of secondary structures especially with N-methylation and side chain hydrogen bond are provided, which can be very useful for further peptide conformation analysis in general.     

Charge-separation reactions of doubly-protonated peptides: Effect of peptide chain length

The collision induced dissociation of doubly-protonated (Ala)_xHis (x=5, 6, 7, 8, 10) peptides have been studied. The major fragmentation reactions observed are symmetrical amide bond cleavages to give the complementary b_m and y_N-m ions, where N is the total number of residues in the peptide. Minor asymmetric cleavage to give doubly-protonated y ions also is observed, involving cleavage near the N-terminus. The shorter peptides (x=5, 6, 7) show major cleavage of the second amide bond to yield b₂ and y_N-2 ions, while (Ala)₁₀His shows major symmetrical cleavage at the fourth and fifth amide bonds. (Ala)₈His appears to be a transitional peptide in showing substantial symmetrical cleavage at the second, fourth, and fifth amide bonds. The results are in general agreement with the bifurcating nature of charge separation noted by Zubarev (J. Am. Soc. Mass Spectrom. 2008, 19, 1755–1763) from a statistical analysis of a large body of doubly-protonated tryptic peptide CID mass spectra. It is shown that the b₂ ion derived from doubly-protonated (Ala)₅His has a protonated oxazolone structure.     

Influence of N-methylation on a cation-pi interaction produces a remarkably stable beta-hairpin peptide

The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.     

N-Nosyl-α-amino acids in solution phase peptide synthesis

A highly efficient and practical synthesis of peptides in solution phase has been developed. The procedure is based on the use of p-nitrobenzenesulfonyl (nosyl) group for the protection of the amino function of α-amino acids. Every step of the procedure, protection of the amino function by the nosyl group, formation of the peptide bond, and removal of the sulfonamide group, is characterized by high yields and excellent purity of the final products. The described strategy allows the preparation of short peptide sequences keeping the chiral integrity of amino acid precursors. Compatibility of nosyl group with the side-chain protecting groups used in Fmoc-based strategy is demonstrated. The method here presented is an alternative strategy that could provide advantages for future peptide synthesis.     

Dissociation of the peptide bond in protonated peptides

The dissociation of the amide (peptide) bond in protonated peptides, [M + H](+), is discussed in terms of the structures and energetics of the resulting N-terminal b(n) and C-terminal y(n) sequence ions. The combined data provide strong evidence that dissociation proceeds with no reverse barriers through interconverting proton-bound complexes between the segments emerging upon cleavage of the protonated peptide bond. These complexes contain the C-terminal part as a smaller linear peptide (amino acid if one residue) and the N-terminal part either as an oxazolone or a cyclic peptide (cyclic amide if one residue). Owing to the higher thermodynamic stability but substantially lower gas-phase basicity of cyclic peptides vs isomeric oxazolones, the N-terminus is cleaved as a protonated oxazolone when ionic (b(n) series) but as a cyclic peptide when neutral (accompanying the C-terminal y(n) series). It is demonstrated that free energy correlations can be used to derive thermochemical data about sequence ions. In this context, the dependence of the logarithm of the abundance ratio log[y(1)/b(2)], from protonated GGX (G, glycine; X, varying amino acid) on the gas-phase basicity of X is used to obtain a first experimental estimate of the gas-phase basicity of the simplest b-type oxazolone, viz. 2-aminomethyl-5-oxazolone (b(2) ion with two glycyl residues).     

Effects of polymer chain length and stiffness on phase separation dynamics of semidilute polymer solution

Three-dimensional dissipative particle dynamics (DPD) simulations were performed to investigate the phase separation dynamics of semidilute polymer solutions with different polymer chain length and stiffness. For the polymer solution composed of shorter and more flexible chains, a crossover of the domain growth exponent from 1/3 to 2/3 was observed during the course of phase separation, indicating that the growth mechanism altered from diffusion to interfacial-tension driven flow. When the chain flexibility was kept the same but the chain was lengthened to allow for the chain entanglement to occur, the growth exponent changed to 1/4 in the diffusion-dominating coarsening regime while the growth exponent remained 2/3 in the flow-dominating regime. When the chain length was kept short but the stiffness was increased, the growth exponent became 1/6 in the diffusion-dominating regime and little effect was observed in the flow-dominating coarsening regime. The slow down of the phase separation dynamics in the diffusion-dominating coarsening could be explained by that the polymer chains could only perform wormlike movement when chain entanglements occurred or when the chain motion was limited by chain stiffness during phase separation. Moreover, when both the effects of chain length and stiffness were enhanced, polymer networks composed of longer and stiffer chains appeared and imposed an energy barrier for phase separation to occur. As a result, the polymer solution with stiffer and longer chains required a larger quench depth to initiate the phase separation and caused the delay in crossover of the coarsening mechanism from diffusion to flow.     

Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.     

Synthesis of lipidated eNOS peptides by combining enzymatic, noble metal- and acid-mediated protecting group techniques with solid phase peptide synthesis and fragment condensation in solution

Lipid-modified proteins play decisive roles in important biological processes such as signal transduction, organization of the cytoskeleton, and vesicular transport. Lipidated peptides embodying the characteristic partial structures of their parent lipidated proteins and semisynthetic proteins synthesized from such peptides are valuable tools for the study of these biological phenomena. We have developed an efficient synthesis strategy that allows for the synthesis of long multiply lipidated peptides embodying various side chain functional groups. The strategy was successfully applied in the synthesis of the N-terminal undetrigintapeptide of endothelial NO-synthase and related lipopeptides. Key elements of the synthesis strategy are the combined use of the enzyme-labile para-phenylacetoxybenzyloxycarbonyl (PhAcOZ) urethane as N-terminal blocking group, the Pd0-sensitive allyl ester as C-terminal protecting function and acid-labile side chain protecting groups for solution-phase synthesis of labile S-palmitoylated building blocks under the mildest conditions with solid-phase techniques and solution-phase fragment condensations. The successful synthesis of the triply lipidated 29-mer eNOS peptide convincingly demonstrates the full capacity of the protecting group methods.     

Correlation of phase equilibria of amino acids and peptides in aqueous solution based on the perturbation theory of equation of state

In this paper, the activity coefficients of amino acids and simple peptides in aqueous solutions were correlated by using a three parameters model based on the perturbation theory. The adjustable parameters of this model were obtained from the experimental data and a relation for calculating the activity coefficient is derived. The calculated activity coefficients of amino acids and simple peptides obtained show that the equation of state based on the perturbation model can be used to correlate the activity coefficients of amino acids more accurately than the other models. A correlation for the solubility of amino acids in aqueous solutions is also derived. The results show that this correlation can accurately correlate the solubility of amino acids in aqueous solution over a wide range of temperatures (0–100 °C).     

Diffusion and phase separation in polymer solution during asymmetric membrane formation

Most of the commercially available polymeric membranes are prepared by the phase inversion process. In this process a thermodynamically stable polymer solution is brought to phase separation by immersing the solution in a surplus of nonsolvent, followed by an exchange of solvent and nonsolvent. The ultimate membrane structure is the result of an interplay of mass transfer and phase separation. Asymmetric membranes as well as symmetrical porous membranes can be obtained. Two types of demixing processes (l-l phase separation and formation of aggregates) can be distinguished by the kinetics of phase separation, as the formation of aggregates is supposed to be a slower process than l-l demixing. Because it is impossible to measure the composition changes during the demixing processes experimentally, a theoretical analysis has to be applied. A suitable formalism to calculate the diffusion induced composition changes in the immersed casting solution, as a function of thermodynamic and hydrodynamic interaction parameters will be described. With this theory it can be shown that two distinctly different mechanisms of membrane formation may occur resulting in two different types of membranes. One type has a relatively thick toplayer and mostly exhibits reverse osmosis, gas separation and pervaporation properties; the other type results in a porous type of membrane, which will exhibit ultra- and microfiltration properties. Model calculations are in agreement with light transmission experiments on membrane forming systems. Therefore, it could be concluded that the elucidation of the diffusion behavior in the immersed polymer film is the key to better understanding of membrane formation by means of immersion precipitation.     

Effects of processing on five selected metals in the durum wheat food chain

The concentration levels of five metals, namely Cd, Cr, Fe, Ni and Pb, were investigated along the durum wheat processing chain, from grain to the final product. Cadmium and Pb are well-known toxic elements and their levels in wheat grain are regulated by the European legislation. Chromium, Fe and Ni were included in the study as markers of metal release from equipment during processing. Durum wheat grain, semolina and pasta were sampled at an industrial plant for milling and pasta making. Samples were taken at different stages along processing in order to elucidate the influence of each stage on the element content. Samples of the water used for grain tempering and dough preparation were also collected. The whole analytical procedure, from sampling to final analytical determinations, was carried out according to a pre-established quality assurance protocol. Analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES).Milling was the key process influencing the concentrations of the studied elements and reduced the metal levels according to a definite element-specific pattern. Purity of the water used as ingredient, element deposition from plant air and metal release from equipment were identified as critical issues in contamination control during processing. In the conditions of this study, it was found that a simple relationship can be established between the original concentrations in durum wheat grain and those in pasta for Cd and Fe, while for Pb, Cr and Ni, a greater uncertainty in the estimation of the levels in the final product is expected.     

Fragmentations of peptide derivatives depending on the positions of proline in negative-ion fast atom bombardment mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters, in which changes in the numbers and positions of prolyl residues were observed, were examined by negative-ion fast atom bombardment mass spectrometry. A significant difference was found among the intensities of the fragment ions formed by cleavage of the benzyloxycarbonyl group, depending on the numbers and positions of prolyl residues in the derivatives. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational differences in the peptide derivatives due to the existence of proline, and permit the prediction of the positions of proline in the peptides.     

Hydroxylation of selected hydrocarbon ions on reaction with methanol in the gas phase

The ion-molecule reactions of selected hydrocarbon cations with methanol which lead to the production of hydroxylated odd-electron molecular ions in the high-pressure ion source of a mass spectrometer or in the central quadrupole of a tandem quadrupole mass spectrometer are described. A wide variety of hydrocarbon cations were investigated, including aliphatic and aromatic cations; of these, only those having a vacant site on an aromatic system appear to undergo the hydroxylation reaction in high yield. A mechanism is proposed for the formation of the molecular ion of phenol from the ion-molecule reaction involving the phenyl cation with methanol. In addition, thermochemical data are provided which support the formation of the postulated species.     

Amino acids and peptides. XXV. Application of newly developed beta-1- and beta-2-adamantylaspartates to peptide synthesis by solid phase and conventional solution methods

Newly developed beta-1- and beta-2-adamantylaspartates [H-Asp(O-1-Ada)-OH and H-Asp(O-2-Ada)-OH] were applied to the synthesis of a C-terminal octapeptide of the beta-subunit of human chorionic gonadotropin (hCG) by a conventional solution method and a hexacosapeptide of the alpha-subunit of insulin receptor (30-55) by a solid-phase method with the objective of suppressing aspartimide formation during the synthesis of aspartylpeptides. The 1-Ada and 2-Ada groups were confirmed to be useful protecting groups for the beta-carboxyl function of the Asp residue.