Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism with LL-37, ATRA-1A, and hBD3-Pep4. In further studies, polymyxin B synergized with LL-37, ATRA-1A, and hBD3-Pep4 while Rifampicin synergized with LL-37 and mCRAMP for intracellular killing of mycobacteria residing inside macrophages. These studies provide the foundation for the potential development of synthetic cationic antimicrobial peptides with activity against mycobacteria.

The thioester surrogate 3,4-diaminobenzoic acid (Dbz) facilitates the efficient synthesis of peptide thioesters by Fmoc chemistry solid phase peptide synthesis and the optional attachment of a solubility tag at the C-terminus. The protection of the partially deactivated ortho-amine of Dbz is necessary to obtain contamination-free peptide synthesis. The reported carbamate protecting groups promote a serious side reaction, benzimidazolinone formation. Herein we introduce the Boc-protected Dbz that prevents the benzimidazolinone formation, leading to clean peptide o-aminoanilides suitable for the total chemical synthesis of proteins.

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a “xenonucleus”, which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

16Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and 17 the pharmaceutical industry, respectively. Here we propose a general design principle for constructing 18 small peptide-based protein-specific folding modifiers. We construct a 'xenonucleus', which is a pre-19folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the 20 folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy 21 transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the 22 ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the 23 refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for 24 constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined 25 contiguous folding nucleus. 26 27 flow kinetics, time resolved Forster Resonance Energy transfer, NMR, and coarse grain simulation 70 studies show that kinetics of ubiquitin folding can indeed be made faster by a suitable xenonucleus 71 interacting at the appropriate site. 72 73 Results: 74 75Refolding kinetics: Ubiquitin has been widely studied for its thermodynamic and mechanical stability. 76 Previous mutational studies on Ubiquitin, especially using the F45W mutant, has established 77 tryptophan fluorescence as a valuable probe for studying its folding. Here, we measured the refolding 78 kinetics of Ubiquitin (F45W) in presence and in absence of a 19 residue nucleus mimic. It has the amino 79 acid sequence C-MQIFVKTLTGKTITLEV-C, which is the same as residues 1 to 17 of Ubiquitin, except for 80 the terminal cysteines. It has a disulphide bridge between the termini, and will henceforth be called 81 the 'stapled xenonucleus'. We use a three syringe stopped-flow fluorescence instrument (SFM300, 82Biologic, see Methods) to change the GdnHCl concentration from 4.25 M to a series of lower 83 concentrations in less than 6 ms. We measure the change in fluorescence intensity of the Trp residue 84as a function of time as a reporter for the progress of folding (or unfolding). The unfolded state of 85 ubiquitin F45W has a higher quantum yield and has an emission maximum at 360 nm, but when it 86 goes to the folded form, the emission blue-shifts to 340 nm. The quantum yield of fluorescence also 87 goes down (it is speculated that a backbone carboxyl oxygen may be quenching the fluorescence) 57,58 . 88Hence, as folding proceeds, the overall fluorescence signal at 360 nm decreases. We have plotted the 89 change in the fluorescence intensity as a function of time for ubiquitin F45W, with (Figure 1 (A), red) 90 and without (Figure 1 (A), blue) the xenonucleus. The data are fitted with a two-component 91 exponential decay function: 92 102 Refolding kinetics as a function of the xenonucleus concentration: We also perform the refolding 103 experiment as a function of the concentration of the xenonucleus peptide...