Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Molecular Detection of Candidatus Orientia chuto in Wildlife, Saudi Arabia.

Scrub typhus is a zoonosis caused by 3 species of Orientia bacteria, including Candidatus Orientia chuto. This species is known only from a human case in Dubai and infections in wildlife in Kenya. We report molecular detection of Candidatus O. chuto in 2 wild rodent species from Saudi Arabia.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

Challenges in the use of sortase and other peptide ligases for site-specific protein modification.

Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.

Deception of Ambient Temperature Does Not Elicit Performance Benefits During a 5 km Run in Hot, Humid Conditions.

Hanson, NJ, Carriveau, DM, Morgan, HE, Smith, AR, Michael, TJ, and Miller, MG. Deception of ambient temperature does not elicit performance benefits during a 5 km run in hot, humid conditions. J Strength Cond Res 32(8): 2250-2257, 2018-The purpose of this study was to investigate the effect of deception of ambient temperature on 5 km performance in recreational runners. Eleven participants (6 men, 5 women) each performed three 5 km runs in a random order consisting of a control trial (CON) in temperate conditions (21° C, 43% RH), a hot humid trial (HOT; 31° C, 65% RH) and a deception trial (DEC; 31° C, 65% RH), where participants were told it was 5° C lower than it actually was. Overall completion time was recorded at the end of trials; thermal sensation (TS), rating of perceived exertion (RPE), and core temperature (TC) were recorded each kilometer. Participants completed the 5 km run faster in the CON condition (23:18 ± 2:05; mean ± SD) compared with DEC (p = 0.005) and HOT (p = 0.014). There was no difference in completion time (p = 0.554) between DEC (25:11 ± 2:41) and HOT (24:25 ± 2:47). Similarly, TS was lower in the CON condition (5.7 ± 0.2) compared with DEC and HOT (p < 0.001 and p = 0.016, respectively) and no differences were seen between the DEC (6.4 ± 0.2) and HOT (6.5 ± 0.2) conditions. No differences in RPE (p = 0.115) or rise in TC (p = 0.289) were seen between the 3 conditions. Deception of the environmental conditions did not positively affect 5 km running performance, and no differences were seen in physiological or psychological variables.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.