Design and Synthesis of a Siderophore-Based Fluorescent Probe and Its Application in Selective Detection of Aeromonas

Amonabactins, the siderophores produced by some pathogenic bacteria belonging to Aeromonas genus, can be used for the preparation of conjugates that can be imported into the cell using their specific transport machinery. Herein, we report the design and synthesis of a new amonabactin-based fluorescent probe by conjugation of the appropiate amonabactin analogue to sulforhodamine B (AMB-SRB) using a thiol-maleimide click reaction. Growth promotion assays and fluorescence microscopy studies demonstrated that AMB-SRB fluorescent probe was able to label the fish pathogenic bacterium A. salmonicida subsp. salmonicida through its outer membrane transport (OMT) protein FstC. The labelling of other Aeromonas species such as the human pathogenic A. hydrophila, indicates that this probe can be a very useful molecular tool for studying the amonabactin-dependent iron uptake mechanism. Furthermore, the selective labelling of A. salmonicida and other Aeromonas species in presence of other fish pathogenic bacteria, suggest the potential application of this probe for detection of Aeromonas in water and other fish farming samples through fluorescence assays.

Surface coat proteins of the potato cyst nematode, Globodera rostochiensis

Summary Potato cyst nematodes (PCN) are estimated to cause over £50 million worth of crop losses in the UK each year. It has been shown that the infective juveniles are able to alter their surface composition to avoid damage from host defence mechanisms. However, relatively few proteins present on the cuticle surface of PCN juveniles have been identified. We have developed a method based upon biotinylation that allows selective labelling of proteins present on the cuticle surface of PCN. Isolated proteins can consequently be affinity purified and identified using mass spectrometry proteomics. Using this technique, we identify a variety of proteins present on the surface of PCN, including all previously described PCN surface proteins. Identification of known surface coat proteins using these methods demonstrates the viability of the process for isolation of novel surface coat proteins. Subsequent analysis confirmed that the genes encoding seven of the novel proteins were expressed in the hypodermis. This work provides a technique for study of surface proteins in a wide range of nematodes and expands our knowledge of the surface proteome of PCN.

Coding of odour and space in the hemimetabolous insect Periplaneta americana

The general architecture of the olfactory system is highly conserved from insects to humans, but neuroanatomical and physiological differences can be observed across species. The American cockroach, inhabiting dark shelters with a rather stable olfactory landscape, is equipped with long antennae used for sampling the surrounding air-space for orientation and navigation. The antennae’s exceptional length provides a wide spatial working range for odour detection; however, it is still largely unknown whether and how this is also used for mapping the structure of the olfactory environment. By selective labelling antennal lobe projection neurons with a calcium sensitive dye, we investigated the logic of olfactory coding in this hemimetabolous insect. We show that odour responses are stimulus-specific and concentration-dependent, and that structurally related odorants evoke physiologically similar responses. By using spatially confined stimuli, we show that proximal stimulations induce stronger and faster responses than distal ones. Spatially confined stimuli of the female pheromone periplanone-B activate sub-region of the male macroglomerulus. Thus, we report that the combinatorial logic of odour coding deduced from holometabolous insects applies also to this hemimetabolous species. Furthermore, a fast decrease in sensitivity along the antenna, not supported by a proportionate decrease in sensillar density, suggests a neural architecture that strongly emphasizes neuronal inputs from the proximal portion of the antenna.Summary statementBy selective labelling the cockroach’s antennal lobe output neurons, we investigated the logic of olfactory coding in a hemimetabolous insect, showing that odour responses are stimulus-specific, concentration-dependent, and preserve information on the spatial structure of the stimulus.

Photoactive yellow protein and its chemical probes: an approach to protein labelling in living cells

Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging.

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Nanopores, both protein and solid-state, are explored as single molecule analytical tools, but using an experimental platform is challenging. Here we show that a commercially available nanopore device, MinION from Oxford Nanopore Technologies (ONT), successfully accomplishes a task challenging for a conventional analytical tool. Specifically the MinION discriminates among 31 nucleotide (nt) long oligoriboadenylates with a single pyrimidine (Py) substitution, when this pyrimidine is tagged/labeled with a bulky group (Osmium tetroxide 2,2’-bipyridine or OsBp). This platform also discriminates between an osmylated Py (Py-OsBp) followed by a purine (Pu) and a Py-OsBp followed by a second Py-OsBp, leading to the conjecture that the bulky tag enables sensing of a two-nucleotide sequence. Two-nucleotide sensing could greatly improve base-calling accuracy in motor enzyme-assisted nanopore sequencing.We attribute the observed discrimination neither to the specific pore protein nor to OsBp, but to the tag’s bulkiness, that leads to markedly slower translocation and “touching” proximity at the pore’s constriction zone, that forces desolvation and reorganization, and enables strong interactions among the nanopore, the tagged pyrimidine, and the adjacent nucleobase. These results constitute proof-of-principle that size-suitable nanopores may be superior to traditional analytical tools, for the characterization of RNA oligos and microRNAs enhanced by selective labelling.

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.

Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel

Small GTPase proteins alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly three RAS isoforms HRAS, KRAS and NRAS, and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is evidence of direct biochemical crosstalk between the functional G-domains of these proteins. The activation potential of a given GTPase is contingent on a co-dependent interaction with nucleotide and a Mg2+ ion, which bind to individual variants via distinct affinities coordinated by residues in the nucleotide binding pocket. Here, we utilize a selective-labelling strategy coupled with real-time nuclear magnetic resonance (NMR) spectroscopy to monitor nucleotide exchange, GTP hydrolysis and effector interactions of multiple small GTPases in a single complex system. We provide new insight on nucleotide preference and the role of Mg2+ in activating both wild-type and oncogenic mutant enzymes. Multiplexing reveals GEF, GAP and effector binding specificity in mixtures of GTPases and establishes the complete biochemical equivalence of the three related RAS isoforms. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately resolve GTPase activation potential, as GTPases such as RALA or the G12C mutant of KRAS demonstrate fast exchange kinetics but have a high affinity for GDP. Further, we propose that the G-domains of small GTPases behave autonomously in solution and nucleotide cycling proceeds independent of protein concentration but is highly impacted by Mg2+ abundance.

Selective labelling of arginine residues engaged in binding sulfatedglycosaminoglycans

IAbstractThe activities of hundreds of proteins in the extracellular space are regulated by binding to the glycosaminoglycan heparan sulfate (HS). These interactions are driven by ionic bonds between sulfate and carboxylate groups on the polysaccharide and the side chains of basic residues in the protein. Here we develop a method to selectively label the guanidino side chains of arginine residues in proteins that engage the anionic groups in the sugar. The protein is bound to heparin (a common experimental proxy for HS) on an affinity column. Arginine side chains that are exposed to solvent, and thus involved in binding, are protected by reaction with the dicarbonyl phenylgyoxal (PGO). Elution of the bound proteins then exposes arginine side chains that had directly engaged with anionic groups on the polysaccharide. These are reacted with hydroxyl-phenylglyoxal (HPG). PGO was found to generate three products: a 1:1 product, the 1:1 water condensed product and a 2:1 PGO:arginine product. These three reaction products and that of HPG had distinct masses. Scripts were written to analyse the mass spectra and so identify HPG labelled arginine residues. Proof of principle was acquired on model peptides. The method was then applied to the identification of heparin binding arginine residues in fibroblast growth factors (FGF) 1 and 2. The data demonstrate that four out of eleven arginine residues on FGF2 and five out of six arginine residues of FGF1 engage heparin. Our approach provides a rapid and reliable means to identify arginines involved in functional complexes such as those of proteins with heparin