translation with d-amino acids

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNAGly that was selected according to the rules of ‘thermodynamic compensation’. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions

Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues.

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the beta-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-nitrophenyl beta-D-glucopyranoside [K(m)=0.27+/-0.02 mM and kcat/K(m)=(2.1+/-0.2)x10(6) M(-1) x s(-1)] and 2,4-dinitrophenyl beta-D-glucopyranoside [K(m)=0.16+/-0.02 mM and k(cat)/K(m)=(2.9+/-0.4)x10(6) M(-1) x s(-1)] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/K(m)) on pH revealed a dependence on a group of pK(a)< or =7.8 in the enzyme-substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside caused time-dependent inactivation (K(i)=2.4+/-0.7 mM and k(i)=0.59+/-0.05 min(-1)) due to the accumulation of a trapped glycosyl-enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (>10(4)), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product

Activity-Dependent Photoaffinity Labeling of Metalloproteases

Metalloproteases, notably members of the matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) families play crucial roles in tissue remodeling, the liberation of growth factors and cytokines from cell membranes (shedding) and cell-cell or cell-matrix interactions. Activity of MMPs or ADAMs must therefore be tightly controlled in time and space by activation of pro-enzymes upon appropriate stimuli and inhibition by endogenous tissue inhibitors of metalloproteases (TIMPs) or α2-macroglobulin to prevent irreversible tissue damage due to excessive degradation or uncontrolled release of potent inflammatory mediators, such as tumor necrosis factor-α (TNF-α).Although there is a wide range of methods to measure the amount of metalloproteases based on immunological approaches, relatively little is known about the activation status of a given enzyme at any given time and location. This information is, however, critical in order to understand the function and possible implication of these enzymes in disease. Since metalloproteases use an active-site bound water molecule to cleave the peptide bond, it is not possible to apply known active-site-directed labeling approaches with electrophilic "warheads." We therefore developed novel metalloprotease inhibitors that contain a photoactivatable trifluoromethylphenyldiazirine group and show that such inhibitors are suitable for activity-dependent photoaffinity labeling of MMPs and ADAMs.</p

Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase s54 and response regulator LipR

Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase s54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins

Quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid by selected reaction monitoring with microfluidics nano-liquid-chromatography-mass spectrometry

Quantitative protein analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; similar to 90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies. (C) 2012 Elsevier B.V. All rights reserved

Activity-Dependent Photoaffinity Labeling of Metalloproteases

Metalloproteases, notably members of the matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) families play crucial roles in tissue remodeling, the liberation of growth factors and cytokines from cell membranes (shedding) and cell-cell or cell-matrix interactions. Activity of MMPs or ADAMs must therefore be tightly controlled in time and space by activation of pro-enzymes upon appropriate stimuli and inhibition by endogenous tissue inhibitors of metalloproteases (TIMPs) or α2-macroglobulin to prevent irreversible tissue damage due to excessive degradation or uncontrolled release of potent inflammatory mediators, such as tumor necrosis factor-α (TNF-α).Although there is a wide range of methods to measure the amount of metalloproteases based on immunological approaches, relatively little is known about the activation status of a given enzyme at any given time and location. This information is, however, critical in order to understand the function and possible implication of these enzymes in disease. Since metalloproteases use an active-site bound water molecule to cleave the peptide bond, it is not possible to apply known active-site-directed labeling approaches with electrophilic "warheads." We therefore developed novel metalloprotease inhibitors that contain a photoactivatable trifluoromethylphenyldiazirine group and show that such inhibitors are suitable for activity-dependent photoaffinity labeling of MMPs and ADAMs