Semisynthesis of Dimeric Proteins by Expressed Protein Ligation

A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a double expressed protein ligation reaction between thioester peptides and a new bis-cysteinyl linker. The protocol was also applied to the synthesis of heterodimers

Tryptophan-PNA gc Conjugates Self-Assemble to Form Fibers

Peptide nucleic acids and their conjugates to peptides can self-assemble and generate complex architectures. In this work, we explored the self-assembly of PNA dimers conjugated to the dipeptide WW. Our studies suggest that the indole ring of tryptophan promotes aggregation of the conjugates. The onset of fluorescence is observed upon self-assembly. The structure of self-assembled WWgc is concentration-dependent, being spherical at low concentrations and fibrous at high concentrations. As suggested by molecular modeling studies, fibers are stabilized by stacking interactions between tryptophans and Watson-Crick hydrogen bonds between nucleobases

Structural Basis of a Temporin 1b Analogue Antimicrobial Activity against Gram Negative Bacteria Determined by CD and NMR Techniques in Cellular Environment

We here report an original approach to elucidate mechanisms of action of antimicrobial peptides and derive crucial structural requirements for the design of novel therapeutic agents. The high resolution structure of TB_KKG6A, an antimicrobial peptide designed to amplify the spectrum of action of Temporin B, bound to <i>E. coli</i> is here determined by means of CD and NMR methodologies. We have also defined, through STD analysis, the residues in closer proximity to the bacterial membrane

NMR characterization of the monomers.

<p>Superimposition of the 2D-TOCSY, expansion of the backbone region, for the g^S and g^OH monomers.</p

Gint4.T-MP conjugate reestablishes LTCC protein levels in HL-1 cardiac cells.

<p>Western Blot analysis for Ca_vα1.2 in total protein lysates from HL-1 cells treated as indicated.</p

Mice infected with GFP-labelled bacteria were analysed using the Leica macrofluo instrument (Wetzlar, Germany) equipped with the Leica application suite 3.1.0 software.

<p>A: liver from mice infected with <i>S. enterica</i> serovar Paratyphi B; B: liver from mice infected with <i>S. enterica</i> serovar Paratyphi B and immediately treated with TA plus TB-YK; C: gastro instestinal tract from mice infected with <i>S. enterica</i> serovar Paratyphi B; D: gastro instestinal tract from mice infected with <i>S. enterica</i> serovar Paratyphi B and immediately treated with TA plus TB.</p

CD spectra of: TB (blue, continuous line), TB-YKβA (red continuous line) in Sodium Phosphate buffer pH 7.4 (-); TB (blue dashed line) and TB-YKβA (red dashed line) in SodiumPhosphate buffer+SDS pH 7.4.

<p>CD spectra of: TB (blue, continuous line), TB-YKβA (red continuous line) in Sodium Phosphate buffer pH 7.4 (-); TB (blue dashed line) and TB-YKβA (red dashed line) in SodiumPhosphate buffer+SDS pH 7.4.</p

Temporins initiate their antibacterial activity by drillings holes in the bacterial membrane.

<p>A: <i>S.aureus</i> A170, untreated; B: <i>S aureus</i> A 170 treated for 10 min with TA (8 µg/ml) plus TB-YK (5 µg/ml) (B); C: <i>S.enterica</i> serovar Paratyphi B untreated; D: <i>S.enterica</i> serovar Paratyphi B treated for 10 min with TA (100 µg/ml) plusTB-YK (4 µg/ml).</p

Gint4.T-MP conjugation.

<p>(A) Strategy for the conjugation of Gint4.T aptamer and MP peptide. (B) Analysis of click chemistry reactions between Gint4.T 3’-propargyl adenosine and MP by 12% acrylamide 7 M urea electrophoresis in two different reaction conditions: 1:1,25 (aptamer:peptide) ratio and 1:3 (aptamer:peptide) ratio. The star indicates the Gint4.T/peptide conjugates. (C) Characterization of the aptamer portion by RT-PCR. Gint4.T and Gint4.T-MP were reverse-transcribed, amplified, and loaded on a 3% agarose gel. The star indicates Gint4-T, which is 53 nt. Neg. Ctl RT and Neg. Ctl PCR refers to the mix of reverse-transcription and PCR without template.</p

Expression levels of cytokine genes in mice infected with <i>S.enterica</i> serovar Paratyphi B (10⁵ CFU/mouse) and 6 days lose treated with temporins (35/75 pool).

<p>Each hystogram represents the mean value measured in 2 mice, each one tested in triplicate.</p