Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints theta by solid state (19)F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles tau and rho from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an alpha-helix, tilted away from the membrane normal by 8 degrees, and assembled as an oligomer. The new (19)F NMR label CF(3)-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.
Antibodies that recognize PTMs of histones play a central role in epigenetic proteomic research. Modification-specific antibodies are employed in chromatin immunoprecipitation, for Western blotting and during the immunoprecipitation steps for MS-based global proteomic analyses. Knowledge about the antibodies' off-target binding is essential for the interpretation of experimental data. To address this challenge we developed a fast and cost efficient system for generating peptide bead arrays. We employed this method to establish a bead-based peptide array containing 384 peptides displaying phosphorylated, acetylated, methylated, and citrullinated N-terminal regions of histones H2A, H2B, H3 and H4 and controls. We profiled the binding of 40 PTM-specific antibodies important for epigenetic proteomic research.
Peptide arrays are useful tools to characterize antibodies, to determine sequence specificities of enzymes, or to find interaction partners to given peptide sequences. One widely-used format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. These SPOT arrays have been used successfully in a broad range of applications since their invention at the beginning of the nineties. The simplicity and robustness of this method along with the fully automated synthesis to generate custom arrays with high peptide densities made the SPOT method popular. A drawback of the SPOT method is the use of large reagent volumes and the limited throughput with only one copy of the library. CelluSpots represent a new method that retains the advantages of the SPOT method but allows the production of hundreds of identical copies on microscope slides for parallel screenings with low sample volumes.
monounsaturated acyl chain. Except for the third series, peptide activity correlated with the first moment of the lateral pressure profile, which is a function of lipid acyl chain structure. In vesicles composed of asymmetric phosphatidylcholines, peptide binding and dye efflux are enhanced compared to symmetric, unsaturated lipids with similar pressure profiles. We attribute this to the entropically more favorable interaction of delta-lysin with partially saturated phospholipids. We find that lipid acyl chain structure has a major impact on the activity of delta-lysin and is likely to be an important factor contributing to the target specificity of amphipathic peptides.
Evaluation of the "ResPep continuous flow synthesizer" with real-time UV-monitoring, automated feedback and heating in solid phase peptide synthesis Automation in assembly of peptides via solid phase synthesis following the Fmoc-strategy [1] is a wellestablished method and commonly used for peptide synthesis today. Mostly, due to length of the peptide, the presence of hydrophobic stretches or sterically hindered amino acids, difficulties during peptide synthesis are sequence inherent.
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