Site-specific Fluorescent Labeling Research Articles

Optimization of N-hydroxysuccinimide ester coupling with aminoallyl-modified RNA for fluorescent labeling

ABSTRACT Site-specific fluorescent labeling of RNA is crucial for obtaining the structural and dynamic information of RNAs by fluorescence techniques. Post-synthetic modification of RNA based on N-hydroxysuccinimide (NHS) coupling reaction is an economic, efficient and simple strategy to introduce fluorophore to samples. However, this strategy are not that frequently used in RNA molecules, and the reported reaction conditions and yields varied among different systems. This study results mainly focused on screening the reaction conditions (reactants concentrations, dimethylsulfoxide concentration, solution conditions, pH and reaction time) between NHS-linked fluorophore and aminoallyl-RNA (aa-RNA) to optimize the yield of fluorescent RNA up to 55%, doubled the initial yield. What’s more, as low as one tenth of fluorescent reagent was used in our protocol compared with the reported protocols, greatly reducing the experimental cost. The protocol can be applied as a general guide potentially for RNA labeling by NHS-ester coupling reaction.

Direct in Vitro Selection of Trans-Acting Ribozymes for Posttranscriptional, Site-Specific, and Covalent Fluorescent Labeling of RNA.

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e., intramolecular reaction) and can then be easily engineered for applications in trans (i.e., in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small-molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity toward its target sequence also in the context of total cellular RNA.

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis.

The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells.

Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code.

The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET.

Influenza A virus surface proteins are organized to help penetrate host mucus.

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus's conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication

The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity.Support or Funding InformationThe Welch Foundation (F‐1684)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping Calcium-Sensitive Regions in GCAPs by Site-Specific Fluorescence Labelling.

Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein-protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.

Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

PurposeConstruction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.ProceduresWe used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.ResultsExpression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.ConclusionsThese results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities.

Multipurpose isothiocyanyl alanine/lysine: Use as solvatochromic IR probes and in site specific labeling/ligation of short peptides

The solvatochromic IR responsivity of small side chain –NCS in two unexplored unnatural amino acids, isothiocyanyl alanine (NCSAla = Ita) and lysine (NCSLys = Itl), without perturbing the conformation is demonstrated in two designed short tripeptide (BocAla-NCSAla-Ala-OMe) and hexapeptide (BocLeu-Val-Phe-Phe-NCSLys-Gly-OMe). Demonstration of site specific fluorescent labeling in both the peptides and ligation type reaction in NCSLys indicates the novelty of these two amino acids as alternative to the available canonical amino acids.

Labeling proteins inside living cells using external fluorophores for microscopy.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Site-Specific Fluorescent Labeling of Argonaute for FRET-Based Bio-Assays.

Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute. Using an archaeal Argonaute variant from Methanocaldococcus jannaschii that closely resembles its eukaryotic counterpart, we site-specifically incorporated fluorescent probes into Argonaute. In this chapter, we first describe how to express archaeal Argonaute with the site-specifically engineered unnatural amino acid para-azido-L-phenylalanine (pAzF) and subsequently describe the coupling of a fluorophore exploiting the unique chemistry of the azide group of pAzF. In the second part of the chapter, we present a methodological approach that probes complex formation between acceptor-labeled archaeal Argonaute and guide and target nucleic acids equipped with a donor fluorophore which ultimately allows single-molecule FRET measurements. Furthermore we describe binding and cleavage assays that report on the functionality of Argonaute-nucleic acid complexes.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28.

MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells. Lin28 protein binds to let-7 miRNA precursors to inhibit their maturation. Herein, we developed a FRET-based, high-throughput screening system to identify small-molecule inhibitors of the Lin28-let-7 interaction. We employed unnatural amino acid mutagenesis and bioorthogonal chemistry for the site-specific fluorescent labeling of Lin28, which ensures the robustness and reliability of the FRET-based protein-miRNA binding assay. Using this direct binding assay, we identified an inhibitor of the oncogenic Lin28-let-7 interaction. The inhibitor enhanced the production of let-7 miRNAs in Lin28-expressing cancer cells and reduced the level of let-7 target oncogene products.

Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation

Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains.

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to 1 Mb from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing, show that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment and the role of programmed changes in chromosome structure in replication control are discussed.

Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling.

In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNAcysAmber from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNAcysAmber with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNAcysAmber, which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.

Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes.

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface-exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane-binding events, Cys mutations were introduced throughout IpaB, allowing site-specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association.

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11–12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4°C, and co-internalization with their receptors at 37°C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.