Method For Site-specific Labeling Research Articles

Fluorescence labeling methods influence the aggregation process of α-syn in vitro differently.

In a previous study, the coexistence of different aggregation pathways of insulin and β-amyloid (Aβ) peptides was demonstrated by correlative stimulated emission depletion (STED) microscopy and atomic force microscopy (AFM). This had been explained by suboptimal proteins labeling strategies that generate heterogeneous populations of aggregating species. However, because of the limited number of proteins considered, the failure of the fluorescent labeling that occurs in a large portion of the aggregating fibrils observed for insulin and Aβ peptides, could not be considered a general phenomenon valid for all molecular systems. Here, we investigated the aggregation process of α-synuclein (α-syn), an amyloidogenic peptide involved in Parkinson's disease, which is significantly larger (MW ∼14 kDa) than insulin and Aβ, previously investigated. The results showed that an unspecific labeling procedure, such as that previously adopted for shorter proteins, reproduced the coexistence of labeled/unlabeled fibers. Therefore, a site-specific labeling method was developed to target a domain of the peptide scarcely involved in the aggregation process. Correlative STED-AFM illustrated that all fibrillar aggregates derived from the aggregation of α-syn at the dye-to-protein ratio of 1 : 22 were fluorescent. These results, demonstrated here for the specific case of α-syn, highlight that the labeling artifacts can be avoided by careful designing the labeling strategy for the molecular system under investigation. The use of a label-free correlative microscopy technique would play a crucial role in the control of the setting of these conditions.

Rational Design of Self-Assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope.

Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade has witnessed the use of a few chemical technologies for this purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox-sensitive SAPs and their systematic self-assembly and disassembly studies using size-exclusion chromatography. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

Preferential light-chain labeling of native monoclonal antibodies improves the properties of fluorophore conjugates

Site specific labeling methods have significant potential to enhance the properties of antibody conjugates. While studied extensively in the context of antibody-drug conjugates (ADCs), few studies have examined the impact of homogenous labeling on the properties of antibody-fluorophore conjugates (AFCs). We report the application of pentafluorophenyl (PFP) esters, which had previously been shown to be reasonably selective for K188 of the kappa light chain of human IgG antibodies, toward producing AFCs. We show that simple replacement of N-hydroxy succinimide (NHS) with PFP dramatically increases the light-chain specificity of near-infrared (NIR) AFCs. Comparing the properties of AFCs labeled using NHS and PFP-activated esters reveals that the latter exhibits reduced aggregation and improved brightness, both in vitro and in vivo. Overall, the use of PFP esters provides a remarkably simple approach to provide selectively labeled antibodies with improved properties.

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3′ and 5′ ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

Site-Specific Labeling of F-18 Proteins Using a Supplemented Cell-Free Protein Synthesis System and O-2-[18F]Fluoroethyl-L-Tyrosine: [18F]FET-HER2 Affibody Molecule.

Although a preparation method for F-18-labeled proteins that used a cell-free translation system and 4-[18F]fluoro-L-proline instead of L-proline has been reported, its introduction depends on amino acid sequences of target proteins. The purpose of the study was to propose site-specific labeling method of F-18 by using cell-free translation systems supplemented with an engineered orthogonal aminoacyl-tRNA synthetase derived from Methanocaldococcus jannaschii (pCNF-RS)/suppressor tRNA (tRNACUAopt) pair, O-2-[18F]fluoroethyl-L-tyrosine ([18F]FET), and template DNA inserted with an amber codon. [18F]FET was prepared from the corresponding precursor and determined whether [18F]FET could be incorporated into an affibody molecule for human epidermal growth factor receptor type 2 (HER2; ZHER2:342) as the 21st amino acid used with the pCNF-RS-tRNACUAopt pair and template DNA inserted with an amber codon in a cell-free translation system. Using SKOV-3 cells, we performed an in vitro binding assay of [18F]FET-ZHER2:342. Furthermore, in vivo positron emission tomography (PET) imaging in SKOV-3 xenograft-bearing mice was performed after the intravenous administration of [18F]FET-ZHER2:342. [18F]FET was successfully incorporated into proteins by using commercially available cell-free protein synthesis reagents with a pCNF-RS-tRNACUAopt pair and template DNA of the desired proteins inserted with an amber codon. The mean radiochemical yield (non-decay-corrected) of [18F]FET-ZHER2:342 was 6.5 ± 4.1%. An in vitro cell binding assay revealed that SKOV-3 cells-bound [18F]FET-ZHER2:342 expressed HER2. The in vivo PET imaging in SKOV-3 xenograft-bearing mice revealed that [18F]FET-ZHER2:342 accumulated in SKOV-3 xenografts. The method proposed in this study might be useful for preparing proteins with F-18 and molecular imaging in the preclinical development.

Cell-Free Synthesis of Site-Specifically Double-Labeled Proteins for More Accurate Single-Molecule FRET Studies

Single molecule FRET (smFRET) is a powerful tool for looking at protein folding and conformational dynamics (1). The application of smFRET to interesting proteins involved in pathologies or drug targeting is limited when the protein of interest is difficult to express in vivo due to toxicity and where cell-free expression is required. Moreover, site-specific labeling is an important issue for smFRET (2), but there is still a lack in simple and robust methods for site-specific labeling of in vitro synthesized proteins for smFRET studies. The conventional method is to double label the protein via cysteine residues (3), but as a consequence of a single chemistry, labeling specificity is limited and would lead to an inaccurate picture of the studied population (4). Incorporation of unnatural amino acids carrying an orthogonal chemistry can increase labeling specificity as it was reported (4). However, described methods were so far mainly considered for smFRET studies performed on in vivo synthesized proteins.By using human calmodulin (hCaM) as a model protein we have developed an alternative method for site-specific labeling of cell-free synthesized proteins. Site-specificity of our system allowed us to obtain sharper FRET histograms compared to the conventional labeling method. In addition, by demonstrating functionality of the labeled hCaM through binding experiments performed in presence of ligands and partners, we have shown the biological relevance of our method.In summary, we have developed a robust and simple method that enables the accurate study of cell-free synthesized proteins in smFRET. This method can be used for studying proteins that cannot be expressed in vivo, as it would be for toxic or membrane proteins.1. Tan, Y. W., J. A. Hanson, J. W. Chu, and H. Yang. 2014. Confocal single-molecule FRET for protein conformational dynamics. Methods Mol. Biol. 1084:51-62.2. Joo, C., and T. Ha. 2012. Labeling proteins for single-molecule FRET. Cold Spring Harb. Protoc. 2012:1009-1012.3. Kim, Y., S. O. Ho, N. R. Gassman, Y. Korlann, E. V. Landorf, F. R. Collart, and S. Weiss. 2008. Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem. 19:786-791.4. Seo, M. H., T. S. Lee, E. Kim, Y. L. Cho, H. S. Park, T. Y. Yoon, and H. S. Kim. 2011. Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid. Anal. Chem. 83:8849-8854.

Toward Adding Complexity in Single Molecule FRET Studies of DNA Mismatch Repair

Mistakes made during DNA replication are targeted for post-replicative repair by the DNA mismatch repair system. Purified reconstitutions of DNA mismatch repair require multiple protein interactions, and in vivo studies of DNA mismatch repair function have identified layers of spatial and temporal regulation. We have previously developed single molecule FRET experiments for studies of purified in vitro studies of DNA mismatch interactions from Thermus Aquaticus MutS and MutL. In this poster we present our recent progress developing single molecule FRET assays for more complex studies of DNA repair. In particular, we highlight our work using unnatural amino acid engineering and other methods for site-specific labeling of yeast repair proteins, detailed analysis of kinetics to reveal nucleotide regulation of protein conformations, and steps toward studies in live cells.

Illuminating biological processes through site-specific protein labeling.

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

Facile Preparation of a Thiol-Reactive (18)F-Labeling Agent and Synthesis of (18)F-DEG-VS-NT for PET Imaging of a Neurotensin Receptor-Positive Tumor.

Accumulating evidence suggests that neurotensin receptors (NTRs) play key roles in cancer growth and survival. In this study, we developed a simple and efficient method to radiolabel neurotensin peptide with (18)F for NTR-targeted imaging. The thiol-reactive reagent (18)F-(2-(2-(2-fluoroethoxy)ethoxy)ethylsulfonyl)ethane ((18)F-DEG-VS) was facilely prepared through 1-step radiofluorination. After high-pressure liquid chromatography purification, (18)F-DEG-VS was incubated with the c(RGDyC) and c(RGDyK) peptide mixture to evaluate its specificity toward the reactive thiol. Thiolated neurotensin peptide was then labeled with (18)F using this novel synthon, and the resulting imaging probe was subjected to receptor-binding assay and small-animal PET studies in a murine xenograft model. The imaging results and metabolic stability of (18)F-DEG-VS-NT were compared with the thiol-specific maleimide derivative N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide-neurotensin ((18)F-FBEM-NT). (18)F-DEG-VS was obtained in high labeling yield. The reaction of (19)F-DEG-VS was highly specific for thiols at neutral pH, whereas the lysine of c(RGDyK) reacted at a pH greater than 8.5. (18)F-DEG-VS-c(RGDyC) was the preferred product when both c(RGDyK) and c(RGDyC) were incubated together with (18)F-DEG-VS. Thiolated neurotensin peptide (Cys-NT) efficiently reacted with (18)F-DEG-VS, with a 95% labeling yield (decay-corrected). The radiochemical purity of the (18)F-DEG-VS-NT was greater than 98%, and the specific activity was about 19.2 ± 4.3 TBq/mmol. Noninvasive small-animal PET demonstrated that (18)F-DEG-VS-NT had an NTR-specific tumor uptake in subcutaneous HT-29 xenografts. The tumor-to-muscle, tumor-to-liver, and tumor-to-kidney ratios reached 30.65 ± 22.31, 11.86 ± 1.98, and 1.91 ± 0.43 at 2 h after injection, respectively, based on the biodistribution study. Receptor specificity was demonstrated by blocking experiment. Compared with (18)F-FBEM-NT, (18)F-DEG-VS-NT was synthesized with fewer steps and provided significantly improved imaging quality in vivo. We have established a facile (18)F-labeling method for site-specific labeling of the Cys-NT. Using this method, we synthesized an NTR-targeted PET agent, which demonstrated high tumor-to-background contrast.

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.

Site-specific labeling of baculovirus in an integrated microfluidic device

Labeling of viruses can be used to reveal viral infection pathways and screen potential anti-viral drugs. Complex procedures, including virus cultivation, purification and labeling are involved in traditional virus labeling methods. And the manipulation of living virus brings risk to researcher health. In this work, we report a general method for site-specific labeling of the envelope virus in an integrated microfluidic device with simple procedures and high security. Site-specific labeling of virus was achieved by fusing the biotin acceptor peptide (AP-tag) and the biotin ligase enzyme (BirA enzyme) with the envelope protein GP64 of baculovirus. The AP-tag could be modified by BirA enzyme to introduce the biotin moiety onto the viral envelope. Western blots and fluorescence colocalization analysis proved that the baculoviruses were biotinylated and labeled with high efficiency. The integrated device incorporated several operation steps including cell seeding, cell culture, cell transfection, virus culture and virus labeling. Since virus biotinylation was achieved during the process of virus cultivation, the complex procedures of virus labeling were simplified in our device. Furthermore the whole process could be completed in the integrated microfluidic device, and direct contact between viruses and researchers could be eliminated in our method, which could greatly reduce the risk to researcher health during living virus labeling.

Construction of Semisynthetic DNA–Protein Conjugates with Phi X174 Gene-A* Protein

DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.

Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry

Toward new biotechnology by genetic alphabet expansion, we developed an efficient site-specific labeling method for large RNA molecules. The combination of unnatural base pair transcription and post-transcriptional modification by click chemistry enables simple RNA labeling with a wide variety of functional groups at desired positions, in a one-pot reaction.

Bio-spectroscopic sensing

Sensing of changes in the spectroscopic properties of organic fluorochromes or nanoparticles (NPs) on their interaction with analytes (targets) can be used for analyte detection. This technique has been extensively investigated and is used widely in the analysis of biological substances because of its high sensitivity and excellent temporal and spatial sampling capability. In some cases, spectroscopic sensing produces signals that are visible to the naked eye. Current topics of investigation in this area include the following: (a) development of in situ spectroscopic traps/sensors for real-time analysis; (b) preparation of materials with long-wavelength features to reduce background signals and biological damage; (c) design of chiral spectroscopic sensors for the analysis of biological chiral compounds; and (d) development of site-specific labeling methods to acquire local information on biomacromolecules. To highlight recent progress in biospectroscopic sensing research, six papers, including one review and five research articles, are published in this special topic. The review by Qian group [1] covers the fabrication method for gold nanoshells and their applications in bio-spectroscopic sensing, and includes many results from their recent studies. The study by Zhao group [2] discusses development of a facile approach for the synthesis of “inert dye” doped fluorescent silica NPs. The prepared NPs are stable and brightly fluorescent in aqueous solution, and have great potential for bio-analysis. Li group [3] developed a homogeneous fluorescence resonance energy transfer (FRET) method for single nucleotide polymorphism assay by integrating rolling circle amplification and cationic conjugated polymers. Yang group [4] reports a highly selective and sensitive fluorescent probe for singlet oxygen based on intramolecular FRET of anthryl appended porphyrins. Tang group [5] designed and synthesized a near-infrared fluorescent probe (BODIPY-Se) to detect F – ions, and found that it could be used to image F –

A Novel Method to Probe Membrane Protein Topology Using Unnatural Amino Acid Mutagenesis and Antibody Epitope Tagging

We have developed a novel strategy to probe the topology of membrane proteins in their native bilayer environment. Our technique relies on unnatural amino acid mutagenesis to incorporate p-azido-L-phenlyalanine at a specific site in the expressed target protein. The reactive azido moiety facilitates Staudinger-Bertozzi ligation chemistry to introduce a monoclonal antibody (mAb) epitope-tagged phosphine derivative. This site-specific labeling method allows the flexibility and precision of single codon scanning, and appears to be superior to current biochemical approaches that rely on chemical modifications and/or introduction of epitope tags by mutagenesis. Our approach can be used to identify gross topological determinants for transmembrane proteins of unknown topology as well as to elucidate secondary structural elements with chemical precision. We demonstrate the experimental feasibility of our technique on human C-C chemokine receptor 5 (CCR5), a heptahelical transmembrane G protein-coupled receptor (GPCR) of known topology. CCR5 is a major co-receptor for HIV-1 entry into host cells. We labeled CCR5 in membranes with FLAG peptide epitope-phosphine at various sites on the receptor's intra- and extracellular surfaces. The differential reactivity of the FLAG epitope-phosphine reagent for the azido group on the various CCR5 mutants correlated to the known topology of CCR5 and defined specific helical boundaries. We further applied the new label/probe technology to mammalian cells in culture in order to label extracellular sites on surface-expressed CCR5. Our new method appears to be satisfactory to probe membrane protein topology in polytopic membrane proteins and has potential applications in the study of receptor signaling mechanisms in live cells.

Förster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method

BackgroundWhile the static structure of the intracellular Ca2+ release channel, the ryanodine receptor type 1 (RyR1) has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes.Methodology/Principal FindingsA novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His) tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni2+/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP). GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm). Cy3NTA bound to His10-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His10 tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His10 tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other.Conclusions/SignificanceThese findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1 structure.

N-Terminal Specific Fluorescence Labeling and its Use in Local Structure Analysis of Proteins (Invited Review)

The function of a protein correlates closely with its structure; even partial structural disorder/misfolding may lead to various diseases such as Parkinsons disease. Therefore, analysis of the local structure of a protein (e.g., detecting conformation changes during folding/unfolding, and obtaining the information on local polarity, viscosity, etc.) is of great importance for proteomics studies, and in this respect fluorescence spectroscopy plays a crucial role because of its great temporal and spatial sampling capability. In particular, fluorescent probes combined with a site-specific labeling technique have been widely used in structural analysis of proteins due to their powerful ability in the elucidation of various properties and functions of proteins. The developments of excellent spectroscopic probes as well as site-specific labeling methods are the prerequisites for conducting such a kind of study. Herein we review the progress and use of N-terminal specific labeling and fluorescence probes in local structure analysis of proteins, including some results of our recent studies on α-lactalbumin, β-lactoglobulin, and a dimeric protein of DsbC. Keywords: N-Terminal specific labeling, fluorescent probes, local structure analysis, proteins

An enzymatic method for site-specific labeling of recombinant proteins with oligonucleotides

DNA was site-specifically conjugated to a substrate peptide of microbial transglutaminase fused to the N- or C-terminus of target proteins without the loss of the proteins' functions of interest.

FlAsH labeling of a nuclear receptor domain (D domain of ultraspiracle) fused to tetracysteine tag

Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains.