Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.

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.

Further assessments of ligase LplA-mediated modifications of proteins in vitro and in cellulo.

BACKGROUND: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. METHODS AND RESULTS: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N3-lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. CONCLUSIONS: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.

Thioester-Assisted Sortase-A-Mediated Ligation.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels.

The use of photoactivatable dyes in STED microscopy has so far been limited by two-photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB-2SiR, a fluorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water-soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.

Diversity and functional characterization of endophytic Methylobacterium isolated from banana cultivars of South India and its impact on early growth of tissue culture banana plantlets.

AIMS: This study aimed at determining the distribution, colonization and growth promoting nature of Methylobacterium spp. in tissue culture banana plantlets. METHODS AND RESULTS: Leaf samples from different field grown banana cultivars were used for Methylobacterium spp., isolation. Metabolic profile and functional characterization for plant growth-promoting traits of the isolates were assessed. The isolates were confirmed using 16S rRNA gene sequencing analysis, which resulted in six distinct species of Methylobacterium namely M. radiotolerans, M. salsuginis, M. thiocyanatum, M. rhodesianum, M. rhodinum and M. populi. Methylobacterium spp. inoculation experiment was conducted under hydroponic system in tissue culture banana plantlets (germ free) with eight selected isolates. A significant increase in growth parameters of Methylobacterium treated plantlets compared to uninoculated control was observed. Methylobacterium salsuginis TNMB03-gfp29 was developed and colonization micrograph was obtained using confocal laser scanning microscopy (CLSM) and scanning electron microscopy in different parts of banana plantlets (root, stem and leaves). CONCLUSION: Field grown banana plants found to harbour diverse endophytic Methylobacterium population. Our finding suggests that endophytic Methylobacterium species may provide significant plant growth promoting compounds/nutrients to the banana plants. The experimental results demonstrated the efficacy of Methylobacterium spp. as a potential bioinoculant and can be exploited as a phyllosphere and rhizosphere based bioinoculant for the initial establishment and growth of tissue culture banana plantlets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study extended our knowledge on the distribution of Methylobacterium spp. in banana plants and endophytic colonization nature of this particular genus in plants. In addition, efficient isolate (M. salsuginis TNMB03) identified in this study may be promoted as bio-inoculants for banana plants after field evaluation.

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

A New Class of NIR-II Gold Nanocluster-Based Protein Biolabels for In†Vivo Tumor-Targeted Imaging.

The design of bright NIR-II luminescent nanomaterials that enable efficient labelling of proteins without disturbing their physiological properties in vivo is challenging. We developed an efficient strategy to synthesize bright NIR-II gold nanoclusters (Au NCs) protected by biocompatible cyclodextrin (CD). Leveraging the ultrasmall size of Au NCs (<2 nm) and strong macrocycle-based host-guest chemistry, the as-synthesized CD-Au NCs can readily label proteins/antibodies. Moreover, the labelled proteins/antibodies enable highly efficient in vivo tracking during blood circulation, without disturbing their biodistribution and tumor targeting ability, thus leading to a sensitive tumor-targeted imaging. CD-Au NCs are stable in the harsh biological environment and show good biocompatibility and high renal clearance efficiency. Therefore, the NIR-II biolabels developed in this study provide a promising platform to monitor the physiological behavior of biomolecules in living organisms.

Quantum Dot Labelling of Tepary Bean (Phaseolus acutifolius) Lectins by Microfluidics.

Lectins are bioactive proteins with the ability to recognize cell membrane carbohydrates in a specific way. Diverse plant lectins have shown diagnostic and therapeutic potential against cancer, and their cytotoxicity against transformed cells is mediated through the induction of apoptosis. Previous works have determined the cytotoxic activity of a Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) and its anti-tumorigenic effect on colon cancer. In this work, lectins from the TBLF were additionally purified by ionic-exchange chromatography. Two peaks with agglutination activity were obtained: one of them was named TBL-IE2 and showed a single protein band in two-dimensional electrophoresis; this one was thus selected for coupling to quantum dot (QD) nanoparticles by microfluidics (TBL-IE2-QD). The microfluidic method led to low sample usage, and resulted in homogeneous complexes, whose visualization was achieved using multiphoton and transmission electron microscopy. The average particle size (380 nm) and the average zeta potential (-18.51 mV) were determined. The cytotoxicity of the TBL-IE2 and TBL-IE2-QD was assayed on HT-29 colon cancer cells, showing no differences between them (p ≤ 0.05), where the LC50 values were 1.0 × 10-3 and 1.7 × 10-3 mg/mL, respectively. The microfluidic technique allowed control of the coupling between the QD and the protein, substantially improving the labelling process, providing a rapid and efficient method that enabled the traceability of lectins. Future studies will focus on the potential use of the QD-labelled lectin to recognize tumor tissues.

Interaction study between HCV NS5A-D2 and NS5B using 19F NMR.

The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a 19F-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (KD) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by 19F NMR.

A Green BODIPY-Based, Super-Fluorogenic, Protein-Specific Labelling Agent.

We report the development of YC23, a novel green BODIPY-based dimaleimide derivative that undergoes a fluorogenic addition reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.

Efficient synthesis of [18 F]FPyME: A new approach for the preparation of maleimide-containing prosthetic groups for the conjugation with thiols.

An improved high yielding radiosynthesis of the known thiol-reactive maleimide-containing prosthetic group1-[3-(2-[18 F]fluoropyridine-3-yloxy)propyl]pyrrole-2,5-dione ([18 F]FPyME) is described. The target compound was obtained by a two-step one-pot procedure starting from a maleimide-containing nitro-precursor that was protected as a Diels-Alder adduct with 2,5-dimethylfurane. Nucleophilic radiofluorination followed by heat induced deprotection through a Retro Diels Alder reaction yielded, after chromatographic isolation, [18 F]FPyME with a radiochemical yield of 20% in about 60 min overall synthesis time. A variety of other [18 F]fluoropyridine based maleimide-containing prosthetic groups should be accessible via the described synthetic strategy.

Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein.

Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study a carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane-spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein-protein interactions between the trimer subunits remained unlabelled, thus revealing their location.

A comparative study of dietary curcumin, nanocurcumin, and other classical amyloid-binding dyes for labeling and imaging of amyloid plaques in brain tissue of 5×-familial Alzheimer's disease mice.

Deposition of amyloid beta protein (Aß) is a key component in the pathogenesis of Alzheimer's disease (AD). As an anti-amyloid natural polyphenol, curcumin (Cur) has been used as a therapy for AD. Its fluorescent activity, preferential binding to Aß, as well as structural similarities with other traditional amyloid-binding dyes, make it a promising candidate for labeling and imaging of Aß plaques in vivo. The present study was designed to test whether dietary Cur and nanocurcumin (NC) provide more sensitivity for labeling and imaging of Aß plaques in brain tissues from the 5×-familial AD (5×FAD) mice than the classical Aß-binding dyes, such as Congo red and Thioflavin-S. These comparisons were made in postmortem brain tissues from the 5×FAD mice. We observed that Cur and NC labeled Aß plaques to the same degree as Aß-specific antibody and to a greater extent than those of the classical amyloid-binding dyes. Cur and NC also labeled Aß plaques in 5×FAD brain tissues when injected intraperitoneally. Nanomolar concentrations of Cur or NC are sufficient for labeling and imaging of Aß plaques in 5×FAD brain tissue. Cur and NC also labeled different types of Aß plaques, including core, neuritic, diffuse, and burned-out, to a greater degree than other amyloid-binding dyes. Therefore, Cur and or NC can be used as an alternative to Aß-specific antibody for labeling and imaging of Aß plaques ex vivo and in vivo. It can provide an easy and inexpensive means of detecting Aß-plaque load in postmortem brain tissue of animal models of AD after anti-amyloid therapy.

Chemical probes for competitive profiling of the quorum sensing signal synthase PqsD of Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa uses the pqs quorum sensing system to coordinate the production of its broad spectrum of virulence factors to facilitate colonization and infection of its host. Hereby, the enzyme PqsD is a virulence related quorum sensing signal synthase that catalyzes the central step in the biosynthesis of the Pseudomonas quinolone signals HHQ and PQS. We developed a library of cysteine reactive chemical probes with an alkyne handle for fluorescence tagging and report the selective and highly sensitive in vitro labelling of the active site cysteine of this important enzyme. Interestingly, only one type of probe, with a reactive α-chloroacetamide was capable of covalently reacting with the active site. We demonstrated the potential of our probes in a competitive labelling platform where we screened a library of synthetic HHQ and PQS analogues with heteroatom replacements and found several inhibitors of probe binding that may represent promising scaffolds for the development of customized PqsD inhibitors as well as a chemical toolbox to investigate the activity and active site specificity of the enzyme.

Labelling Matrix Metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of zinc-containing proteases that participate in many physiological and pathological processes in vivo. Recently, the MMP network has been established according to a deeper understanding of its functions. Some MMPs have been also regarded as biomarkers of various diseases, including inflammation, nerve diseases, and cancers. MMP labelling has been thus paid more attention in recent decades. Accordingly, both reagents and technologies for MMP labelling have been rapidly developed. Here we summarize the recent development of some MMP labelling methods. This review was identified through keyword (MMPs; labelling; etc.) searches in the ScienceDirect database, Scifinder, Web of Science, and PubMed for which typical cases were used for an inductive overview. In spite of the advances in MMP labelling, selective labelling of a specific MMP is still an open issue. We hope that this article can be helpful in developing specific MMP labelling methods in future.

First thermostable CLIP-tag by rational design applied to an archaeal O6 -alkyl-guanine-DNA-alkyl-transferase.

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6 -benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8 . The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6 -alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant.

Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases.

Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics.

Fluorescent chameleon labels for bioconjugation and imaging of proteins, nucleic acids, biogenic amines and surface amino groups. a review.

Chameleon labels (ChLs) possess the unique property of changing (visible) color and fluorescence on binding to amino groups of biomolecules. MostChLs react with primary aliphatic amino groups such as those in lysine or with amino groups artificially introduced into polynucleic acids or saccharides, but someothers also react with secondary amino groups. Under controlled circumstances, the reactions are fairly specific. The review is subdivided into the following sections: (1) An introduction and classification of fluorescent labels; (2) pyrylium labels that undergo shortwave color changes upon labelling, typically from blue to red; (3) polymethine type of labels (that also undergo shortwave color changes, typically from green to blue; (4) various other (less common) chromogenic and fluorogenic systems; (5) hemicyanine labels that undergolongwavecolor changes, typically from yellow to purple; (6) the application of ChLs to labeling of proteins and oligonucleotides; (7) applications to fluorometric assays and sensing; (8) applications to fluorescence imaging of biomolecules; (9) applications in studies on affinity interactions (receptor-ligand binding); (10) applications in surface and interface chemistry; and (11) applications in chromatography, electrophoresis and isotachophoresis of biomolecules.

[Preparation of luminescent silica nanoparticles with immobilized metal ion affinity for labeling phosphorylated proteins in Western Blot].

Protein phosphorylation is an important type of post-translational protein modification. In Western Blot experiment, the assay of phosphoproteins need special phospho antibodies, which are expensive, difficult to preserve, poorly reproducible. To this end, the immobilized metal ion affinity luminescent silica nanoparticles for instead of phospho antibodies were prepared. A layer of polymer was created on the surface of the silica nanoparticles via co-polymerization to protect the nanoparticles and to functionalize them with the immobilized metal ion affinity property to specifically label the phosphorylated proteins in Western Blot assays. The affinity luminescent silica nanoparticles were prepared with the following procedure. First, the sol-gel precursor fluorescein isothiocyanate-3-aminopropyltriethoxysilane (FITC-APTES) with the fluorescent moiety was prepared by modifying APTES with FITC. The luminescent silica nanoparticles (FITC@SiO2) were synthesized using the Stöber synthesis method in a reversed microemulsion. Briefly, 123.2 mL of cyclohexane, 25.6 mL of n-hexanol, and 5.44 mL of deionized water were ultrasonically mixed, and then 28.3 g of Triton X-100 were added and the mixture was magnetically stirred for 15 min to form a clear and transparent microemulsion system. Within 10 min, 0.8 mL of FITC-APTES precursor, 1.6 mL of tetraethoxysilane (TEOS), and 0.96 mL of concentrated ammonia (25%-27%, mass fraction) were added to the microemulsion, and the mixture was stirred at 24 ℃ for 24 h. After the reaction, the microemulsion system was destroyed by adding 200 mL of ethanol. The resulting FITC@SiO2 luminescent silica nanoparticles were centrifuged, and washed three times with ethanol. After dryness, the FITC@SiO2 nanoparticles were modified with methacryloxy-propyltrimethoxysilane (MPS) to introduce the double bonds for further modification. The functional monomer nitrilotriacetic acid (NTA) and glycidyl methacrylate (GMA) were copolymerized on the surface of the nanoparticles to convert FITC@SiO2-MPS to FITC@SiO2-MPS-GMA-NTA. The polymer coating of the silica nanoparticles was not only able to protect the silica from hydrolysis, but also to introduce the functional groups of nitrilotriacetic acid, which can chelate with metal ions. Elemental analysis demonstrated that the NTA groups had been bonded to the surface of the nanoparticles via copolymerization. The polymerization did not affect the morphology and fluorescence properties of the nanoparticles. The FITC@SiO2-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr4+, Fe3+, and Ti4+, for the enrichment of phosphorylated peptides derived form α-casein tryptic digestion. HPLC-MS analysis indicated that the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles are the best for the enrichment of phosphorylated peptides. The FITC@SiO2-MPS-GMA-NTA-Ti4+ nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment. The electrophoretic band of α-casein could be clearly labeled with the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles, while the bovine albumin band could not be labelled. This indicates that the luminescent FITC@SiO2-MPS-GMA-NTA-Ti4+nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.