SciCombinator

Discover the most talked about and latest scientific content & concepts.

Journal: Chembiochem : a European journal of chemical biology

37

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.

Concepts: DNA, Gene, Signal transduction, Enzyme, Protein folding, Escherichia coli, Biotechnology, Egg white

28

An Abl label: The design of sensors to monitor the activity state of specific protein kinases is challenging due to the complexity of eukaryotic kinomes. Here we describe a peptide-based photoaffinity probe that specifically labels the active conformation of the Abl tyrosine kinase.

Concepts: Amino acid, Signal transduction, Adenosine triphosphate, Enzyme, Kinase, Protein kinase, Phosphorylation, Label

27

We report the synthesis and photolytic properties of caged inorganic phosphates (Pi compounds) based on the 2-(4'-{bis[2-(2-methoxyethoxy)ethyl]amino}-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (EANBP) and 7-(diethylamino)coumarin-4-yl]methyl (DEACM) protecting groups. The EANBP-Pi showed unprecedented photolysis efficiency at 405 nm, with 95 % release of free phosphate and a quantum yield of 0.28. Thanks to the high two-photon sensitivity of the EANBP chromophore, Pi release through two-photon photolysis is also possible, with an action cross section of 20.5 GM at 800 nm. Two bioactivatable acetoxymethyl protection groups were added to the “caged-Pi” compounds. The resulting triesters of phosphoric acid were able to diffuse through the cellular membranes of plant cells. Once inside a cell, the cleavage of these biocleavable motifs by intracellular esterases allows intracellular accumulation of EANBP-Pi. Bis(AM)-EANBP-Pi therefore represents a very attractive tool for study of the Pi signal transduction cascade in living cells.

Concepts: Protein, Organism, Cell membrane, Cell biology, Phosphate, Phosphoric acid, Phosphorus, Transfection

27

Complex bouquet: A recently developed method for trace analyses combining the advantages of GC-MS and (13) C NMR spectroscopy was applied to investigate the volatiles of Penicillium roqueforti. Besides the main compound, aristolochene, several side products of aristolochene synthase and downstream oxidation products en route to PR toxin were identified, giving insight into the biosynthetic pathway.

Concepts: Scientific method, Amino acid, Adenosine triphosphate, Fungus, Vitamin, Protein biosynthesis, Penicillium roqueforti, Aristolochene

27

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.

Concepts: Photosynthesis, Metabolism, Nutrition, Biochemistry, Carbohydrate, Carbohydrates, Sugar, Glycomics

27

New human β-glucocerebrosidase (GCase) ligands with rigid 1,6-anhydro-β-L-idonojirimycin cores have been designed with the aid of molecular modeling. Efficient pharmacological chaperones for the L444P (trafficking-incompetent) mutant GCase enzyme associated with type 2 and 3 Gaucher disease (GD) were identified.

Concepts: Molecular biology, Gaucher's disease

27

Aptamers are single-stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer-based biosensors are prone to producing false-positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false-positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA-DNA duplexes with 1) a quencher-labelled DNA strand (Q1 DNA), 2) a dual-fluorophore-labelled DNA strand (F1 DNAF2 ) and 3) another quencher-labelled DNA strand (Q2 DNA). The addition of a target releases Q2 DNA from the duplex assembly, and produces the expected positive signal from F2 . However, the authenticity of target recognition is validated only if no signal is generated from F1 . We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high-quality detection when RNA aptamers are employed as molecular-recognition elements.

Concepts: DNA, Protein, Gene, Molecular biology, RNA, Molecule, Nucleotide, Aptamer

27

New assembly line, new compound: SIA7248, a new symmetric macrolide, was isolated from a marine-derived Streptomyces strain. Bioinformatic analyses of the identified biosynthetic gene cluster (sia) for SIA7248 suggested a polyketide biosynthesis utilizing an iteratively trans-acting ketoreductase (KR). We characterized SiaM as a trans-KR to catalyse reductions of various β-ketoacyl-thioesters with D-stereospecificity.

Concepts: Amino acid, Molecular biology, Assembly line, Biosynthesis, Macrolide, Polyketide, Malonyl-CoA

26

Nudicaulins are unique alkaloids responsible for the yellow color of the petals of some papaveraceaous plants. To elucidate the unknown biosynthetic origin of the skeleton, a (13) CO2 -pulse/chase experiment was performed with growing Papaver nudicaule plants. (13) C NMR analysis revealed more than 20 multiple (13) C-enriched isotopologues in nudicaulins from the petals of (13) CO2 -labeled plants. The complex labeling pattern was compared with the isotopologue composition of a kaempferol derivative that was isolated from petals of the same (13) CO2 -labeled plants. The deconvolution of the labeling profiles indicated that the nudicaulin scaffold is assembled from products or intermediates of indole metabolism, the phenylpropanoid pathway, and the polyketide biosynthesis. Naringenin-type compounds and tryptophan/tryptamine are potential substrates for the condensation reaction finally generating the aglycone skeleton of nudicaulins.

Concepts: Photosynthesis, Amino acid, Metabolism, Color, Isotope, Yellow, Primary color, Papaver nudicaule

25

Formylglycine-generating enzymes (FGEs) catalyze O2 -dependent conversion of specific cysteine residues of arylsulfatases and alkaline phosphatases into formylglycine. The ability also to introduce unique aldehyde functions into recombinant proteins makes FGEs a powerful tool for protein engineering. One limitation of this technology is poor in vitro activity of reconstituted FGEs. Although FGEs have been characterized as cofactor-free enzymes we report that the addition of one equivalent of Cu(I) increases catalytic efficiency more than 20-fold and enables the identification of stereoselective CH bond cleavage at the substrate as the rate-limiting step. These findings remove previous limitations of FGE-based protein engineering and also pose new questions about the catalytic mechanism of this O2 -utilizing enzyme.

Concepts: Protein, Metabolism, Enzyme, Catalysis, Ribozyme, Chemical equilibrium