Real-time monitoring of solid-phase peptide synthesis using a variable bed flow reactor

On-resin aggregation and incomplete amide bond formation are major challenges for solid-phase peptide synthesis that are difficult to be monitored in real-time. Incorporation of a pressure-based variable bed flow reactor into an automated solid-phase peptide synthesizer permitted real-time monitoring of resin swelling to determine amino acid coupling efficiency and on-resin aggregation

Catalytic RNA and synthesis of the peptide bond

We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested

Role of Peptide Backbone Conformation on Biological Activity of Chemotactic Peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C α,α disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II β-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met- Ac6c -Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended β-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The ϕ and ψ values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel β conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor

The action of rennin on B-casein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University

A study was made of the action of the enzyme rennin on β-casein. Hydrolysis of β-casein initially at a single sensitive bond under controlled conditions of temperature, pH and relative enzyme and substrate concentrations, formed the basis of the investigation. Information on the hydrolysis of this sensitive bond was gained from the isolation of a small peptide produced and from a study of the effect of several parameters on the rate of hydrolysis. Evidence obtained from electrophoresis and gel filtration allowed the assumption that attack on the sensitive bond resulted in a macropeptide and a small peptide of molecular weight about 2000. The small peptide was isolated and partially characterised. As a result it appears that the small peptide is derived from the C-terminal end of the β-casein molecule. A polyacrylamide electrophoresis technique was used to study the effect of ionic strength and calcium ions on the rate of hydrolysis and the rate of appearance and disappearance of degradation products at 10°, 25° and 37°C. It was found that an increase in ionic strength retarded the reaction and the addition of calcium ions at a constant ionic strength further retarded the reaction. Also, the rate of appearance and disappearance of degradation products was found to increase with increasing temperature. A development of the polyacrylamide technique into a quantitative one enabled the determination of the Michaelis constant at pH 6.50 and 37°C for the rennin hydrolysis of β-casein as 9-59 g/1. This technique was also used to study the rate of hydrolysis at pH 6.12, 6.50 and 6.94 where an optimum rate occurred at pH 6.50. Finally, assuming that the small peptide is derived from the C-terminal end of the β-casein molecule and allowing for the sequential degradation elucidated by the temperature studies, alternative courses of rennin degradation of the β-casein molecule have been proposed

Design and synthesis of amidine-type peptide bond isosteres: application of nitrile oxide derivatives as active ester equivalents in peptide and peptidomimetics synthesis.

Amidine-type peptide bond isosteres were designed based on the substitution of the peptide bond carbonyl (C=O) group with an imino (C=NH) group. The positively-charged property of the isosteric part resembles a reduced amide-type peptidomimetic. The peptidyl amidine units were synthesized by the reduction of a key amidoxime (N-hydroxyamidine) precursor, which was prepared from nitrile oxide components as an aminoacyl or peptidyl equivalent. This nitrile oxide-mediated C-N bond formation was also used for peptide macrocyclization, in which the amidoxime group was converted to peptide bonds under mild acidic conditions. Syntheses of the cyclic RGD peptide and a peptidomimetic using both approaches, and their inhibitory activity against integrin-mediated cell attachment, are presented

Local electronic structure of the peptide bond probed by resonant inelastic soft X-ray scattering.

The local valence orbital structure of solid glycine, diglycine, and triglycine is studied using soft X-ray emission spectroscopy (XES), resonant inelastic soft X-ray scattering (RIXS) maps, and spectra calculations based on density-functional theory. Using a building block approach, the contributions of the different functional groups of the peptides are separated. Cuts through the RIXS maps furthermore allow monitoring selective excitations of the amino and peptide functional units, leading to a modification of the currently established assignment of spectral contributions. The results thus paint a new-and-improved picture of the peptide bond, enhance the understanding of larger molecules with peptide bonds, and simplify the investigation of such molecules in aqueous environment

Oberflächen-Plasmon-Resonanz- spektroskopische Studie an verschiedenen Biosensoroberflächen. Charakterisierung von Protein-Peptid und Protein-Lipid Wechselwirkungen

Surface plasmon resonance spectroscopy (SPR spectroscopy) is a technique for the time-resolved measurement of interactions between macromolecules. For thesemeasurements, it is necessary to immobilize a binding partner on the surface of asensor chip. The measurements are recorded as sensorgrams. In the present study,different surfaces were tested. The biological model system for the investigation ofinteractions was the binding of calmodulin to a synthetic peptide which correspondsto the amino-acid sequence of the CaM binding site from the typed nitrogen oxidesynthase. For binding CaM to this peptide, NOS-I WT, the rate constants (k$_{a}$, k$_{d}$) andthe equilibrium constants (KD) were determined by different evaluation methods.The dissociation constants determined from the sensorgrams for the different sensorchip surfaces increased with rising immobilization density. An analysis of the rateconstants determined from the sensorgrams showed that especially k$_{a}$ decreasedsubstantially with increasing immobilization density. The sensorgrams on the differentsensor surfaces could no longer be explained by a monoexponential bond model athigh immobilization density. This was also reflected in the decreasing stoichiometryof peptide NOS-I WT for CaM at increasing immobilization density of the peptide. Thebest agreement with the literature values achieved on a dextran surface at very lowimmobilization density (< 5.8 fmol/mm$^{2}$) on peptide NOS-I WT (K$_{D}$ values of 0.8 - 3.9nM).Furthermore, sensor chips with hydrophobic surfaces were produced to build upheterobilayers or phospholipid bilayers. These sensor chips were used to investigatethe Ca$^{2+}$-dependent membrane association of the Ca$^{2+}$ binding protein recoverin. AnN-terminal myristoyl group anchors recoverin at high calcium concentrations in themembrane. Mutants of recoverin were examined, whose affinities for Ca2+ ions weremodified in comparison to the wild type. All forms of recoverin (native, recombinantlymyristoylated (WT), EF+4, EF-3, EF-2) show a bond to the hydrophobic sensor chipsurface in the presence of Ca$^{2+}$ ions, although with different affinities (EC$_{50}$: nativerecoverin = 18 $\mu$M; WT = 5 $\mu$M; EF+4 = 29 $\mu$M; EF-3 = 8 $\mu$M; EF-2 = 9 $\mu$M).Interestingly enough, it was also possible to detect a bond to the phospholipid layerin the absence of Ca$^{2+}$ which, however, occurred with a markedly lower amplitude

A novel method of analyzing proline synonymous codons in E. coli

AbstractProline is a special imino acid in protein and the isomerization of the prolyl peptide bond has notable biological significance and influences the final structure of protein greatly, so the correlation between proline synonymous codon usage and local amino acid, the correlation between proline synonymous codon usage and the isomerization of the prolyl peptide bond were both investigated in the Escherichia coli genome by using a novel method based on information theory. The results show that in peptide chain, the residue at the first position C-terminal influences the usage of proline synonymous codon greatly and proline synonymous codons contain some factors influencing the isomerization of the prolyl peptide bond

The nonplanarity of the peptide group: Molecular dynamics simulations

The general properties of the peptide bond can be described from a linear combination of two states: a single bond neutral form and a double bond zwitterionic form. However, environmental effects can shift the balance of the linear combination. This would cause the rigidity of torsional rotations of the peptide bond to be environmentally dependent and, in fact, an analysis of protein structures in the protein data bank reveals a different degree of nonplanarity for different secondary structure elements. A potential is presented in which the peptide bond is treated as a linear combination of two states; the coefficients of the two states are updated as the simulation progresses using an extended Lagrangian formalism. The model is applied to the helix/coil transition of polyalanine. Fluctuations in the planarity of the peptide dihedral angle are found to increase the rate constant for the coil to helix transition by a factor of tw

Glutathione as a Prebiotic Answer to alpha-Peptide Based Life.

The energetics of peptide bond formation is an important factor not only in the design of chemical peptide synthesis, but it also has a role in protein biosynthesis. In this work, quantum chemical calculations at 10 different levels of theory including G3MP2B3 were performed on the energetics of glutathione formation. The strength of the peptide bond is found to be closely related to the acid strength of the to-be N-terminal and the basicity of the to-be C-terminal amino acid. It is shown that the formation of the first peptide activates the amino acid for the next condensation step, manifested in bacterial protein synthesis where the first step is the formation of an N-formylmethionine dipeptide. The possible role of glutathione in prebiotic molecular evolution is also analyzed. The implications of the thermodynamics of peptide bond formation in prebiotic peptide formation as well as in the preference of alpha- instead of beta- or gamma-amino acids are discussed. An empirical correction is proposed for the compensation of the error due to the incapability of continuum solvation models in describing the change of the first solvation shell when a peptide bond is formed from two zwitterions accompanied by the disappearance of one ion pair