Exploration of disorder in protein structures by X-ray restrained molecular dynamics

Conformational disorder in crystal structures of ribonuclease-A and crambin is studied by including two independent structures in least-squares optimizations against X-ray data. The optimizations are carried out by X-ray restrained molecular dynamics (simulated annealing refinement) and by conventional least-squares optimization. Starting from two identical structures, the optimizations against X-ray data lead to significant deviations between the two, with rms backbone displacements of 0.45 A for refinement of ribonuclease at 1.53 A resolution, and 0.31 A for crambin at 0.945 A. More than 15 independent X-ray restrained molecular dynamics runs have been carried out for ribonuclease, and the displacements between the resulting structures are highly reproducible for most atoms. These include residues with two or more conformations with significant dihedral angle differences and alternative hydrogen bonding, as well as groups of residues that undergo displacements that are suggestive of rigid-body librations. The crystallographic R-values obtained are approximately 13%, as compared to 15.3% for a comparable refinement with a single structure. Least-squares optimization without an intervening restrained molecular dynamics stage is sufficient to reproduce most of the observed displacements. Similar results are obtained for crambin, where the higher resolution of the X-ray data allows for refinement of unconstrained individual anisotropic temperature factors. These are shown to be correlated with the displacements in the two-structure refinements.     

Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide

The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy. Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol. Six microorganisms, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, P. putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing. However, electroporation of Hok (200 microgml(-1)) into E. coli cells showed a dramatic reduction (100000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells. Electroporation of Hok into P. putida was also very effective with a 500-fold reduction in electrocompetent cells (100 microgml(-1)). Heat shock in the presence of Hok (380 microgml(-1)) resulted in a 5-fold reduction in E. coli cells but had no effect on B. subtilis. In addition, three Hok fragments (Hok(1-28), Hok(31-52) and Hok(16-52)) killed cells when electroporated into E. coli at 200 microgml(-1) (over 1000-fold killing for Hok(1-28), 50-fold killing for Hok(16-52) and over 1000-fold killing for Hok(31-52)). E. coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell.     

Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members prepare cyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified:(i) N-terminal guanidine formation; (ii) C-terminal piperidyl amide formation; and (iii) a novel C-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.     

Formation and characterization of a single Trp-Trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity

Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and beta-structured cationic peptides, because five of indolicidin's 13 residues are tryptophans: H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH(2). Solid phase synthesis of indolicidin gave rise to a minor byproduct that possessed unusual fluorescence and UV absorbance properties compared with authentic indolicidin. The byproduct was purified by combined ion exchange and reversed phase high pressure liquid chromatography steps and was shown be identical to authentic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass analysis of the byproduct revealed a 2-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to form an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed the nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edman degradation revealed that Trp-6 and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digestion with trypsin and chymotrypsin, suggesting that the ditryptophan stabilizes a subset of molecular conformations that are protease resistant but that are absent in the native structure.     

Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

Summary The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members preparecyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified: (i)N-terminal guanidine formation; (ii)C-terminal piperidyl amide formation; and (iii) a novelC-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.