Biarsenical Dye Research Articles

Cross-Strand Split Tetra-Cys Motifs as Structure Sensors in a β-Sheet Protein

We have designed "split tetra-Cys motifs" that bind the biarsenical fluorescein dye 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) across strands of a model beta-rich protein. Our strategy was to divide the linear FlAsH binding tetra-Cys sequence such that dye could be fully liganded only when the strands were arranged in space correctly by native protein conformational proximities. We introduced pairs of alternating cysteines on adjacent beta strands of cellular retinoic acid binding protein to create FlAsH binding sites in the native structure. Selective labeling occurred both in vitro and in vivo relative to sites with fewer than four Cys or with inappropriate geometry. Interestingly, two of the split tetra-Cys motif-carrying proteins bound FlAsH whether native or urea unfolded, while one was capable of binding FlAsH only when native. This latter design exemplifies the potential of split motifs as structure sensors.

Biarsenical−Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.

Resolution of de novo HIV production and trafficking in immature dendritic cells

The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus of HIV p17 matrix within the HIV gag protein. Using this construct in combination with biarsenical dyes, we observed restricted staining of the dTCM to de novo-synthesized uncleaved gag in the DC cytosol. Co-staining with HIV gag antibodies, reactive to either p17 matrix or p24 capsid, preferentially stained mature virions and thus allowed us to track the virus at distinct stages of its life cycle within DCs and upon transfer to neighboring DCs or T cells. Thus, in staining HIV gag with biarsenical dye system in situ, we characterized a replication-competent virus capable of being tracked preferentially within infected leukocytes and observed in detail the dynamic nature of the HIV production and transfer in primary DCs.

The Biarsenical Dye Lumio™ Exhibits a Reduced Ability to Specifically Detect Tetracysteine-Containing Proteins Within Live Cells

Investigating the localisation of proteins within live cells via fluorescence microscopy typically involves the fusion of the protein of interest to a large fluorescent protein such as green fluorescent protein (GFP). Alternate fluorescent labelling technologies such as the fluorescent biarsenical dye molecules (e.g. FlAsH, ReAsH) are preferable to the use of large fusion proteins in many respects and allow greater flexibility in terms of the location of the labelling site. We assessed the ability of the FlAsH-derived biarsenical dye molecule Lumio to label a range of tetracysteine containing proteins within live cells and report that although in some circumstances Lumio is capable of positively detecting such proteins, the sensitivity and specificity of labelling is significantly reduced, making the Lumio-labelling system unsuitable for the detection of a wide range of protein within live cells.

A Red Cy3-Based Biarsenical Fluorescent Probe Targeted to a Complementary Binding Peptide

We have synthesized a red biarsenical fluorescent probe, AsCy3, with good photostability, low pH sensitivity, high absorbance, and good quantum yield. It is directed specifically to a small tetracysteine peptide binding motif, Cy3TAG (CysCysLysAlaGluAlaAlaCysCys), in the presence of other tetracysteine tags. This new probe provides a FRET partner to biarsenical dye FlAsH, making this discovery an important step toward a whole toolkit of colored probes directed to different small peptide motifs.

Characterization of a CFTR construct with a C‐terminal tetracysteine sequence and its use in the visualization of trafficking pathways

Membrane permeable bi-arsenical dyes, such as fluorescein arsenical hairpin (FlAsH), bind tetracysteine-tagged-proteins emitting fluorescence in-vivo. This fluorescence method (developed by R. Tsien) provides a tool to evaluate trafficking pathways in living cells. We generated a tetracysteine-tagged Cystic Fibrosis Transmembrane Regulator (CFTR) protein and studied its expression with the long-term goal of using this protein to study its trafficking during endocytosis. The tetracysteine-tagged CFTR (CFTR-TC) was predominantly expressed in both BHK and in Cos-7 cells as the immature core-glycosylated form (band B) rather than the mature glycosylated band C. Iodide efflux assays of CFTR-TC's function as a chloride channel at the cell surface revealed an insignificant response after cAMP stimulation, likely reflecting its low band C expression. Interestingly, 1 hour FlAsH pre-treatment evoked a mean response difference of −16.0 mV versus untreated (one-way ANOVA p<0.05), suggesting increased band C expression. Our results suggest that FlAsH rescues the surface expression of CFTR-TC and studies are ongoing to visualize its localization and the regulation of its trafficking by proteins involved in the endocytic pathway. The work is supported by the Natural Sciences and Engineering Research Council (postgraduate scholarship) and the Canadian Cystic Fibrosis Foundation BREATHE Program.

Improved Photostable FRET-Competent Biarsenical−Tetracysteine Probes Based on Fluorinated Fluoresceins

We have developed fluoro-substituted versions of the biarsenical-tetracysteine label FlAsH, exhibiting significant improvements in important properties over the original fluorescein derivative. In complexes with tetracysteine targets, F2FlAsH exhibits 50 times improved photostability, lower pH sensitivity, higher absorbance and quantum yield than FlAsH, and F4FlAsH adds a new color to the palette of biarsenical dyes. The two probes also provide a new FRET pair with a larger Ro value (54 A) than any previously obtained with biarsenical dyes.

Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity

Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to beta-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.

Aggregation of a Slow-Folding Mutant of a β-Clam Protein Proceeds through a Monomeric Nucleus

Mechanistic understanding of protein aggregation, leading either to structured amyloid fibrils or to amorphous inclusion body-like deposits, should facilitate the identification of potential therapeutic intervention strategies for the devastating amyloid-based diseases. Here we focus on the in vitro aggregation of a slow-folding mutant of the beta-clam protein, cellular retinoic acid-binding protein I (P39A CRABP I), which forms inclusion bodies when expressed in Escherichia coli. Aggregation was monitored by observing the fluorescence of a fluorescein-based biarsenical dye (FlAsH) that ligates to a tetra-Cys motif, here incorporated into a flexible Omega-loop. The fluorescence signal of FlAsH on the tetra-Cys-containing P39A CRABP I is sensitive to whether this protein is native or unfolded, and was used in combination with other techniques to follow aggregate formation. The aggregation time course is compatible with a nucleation-dependent polymerization model, and detailed kinetic analysis showed that the energetically unfavorable nucleus is monomeric. A similar conclusion was reached previously for poly(Gln) species [Chen, S., Ferrone, F. A., and Wetzel, R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889] and points to an unfavorable equilibrium between the misfolded intermediate and the bulk pool of monomers as causative in aggregation. The P39A mutation, which removes a helix-stop signal, may slow closure of the beta-barrel in P39A CRABP I relative to the wild type, leaving it vulnerable to aggregation. Wide-angle X-ray scattering showed that the amorphous aggregates formed by the aggregation-prone intermediates of P39A CRABP I contain predominantly beta-strands structured in a lamellar fashion with 10.03 A spacing between adjacent beta-sheets.

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregationprone mutant (FlAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.

Genetically targeted chromophore-assisted light inactivation.

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.

Imaging Protein Interactions and Gene Expression in Individual Cells by Fluorescence Resonance Energy Transfer

Abstract Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway. A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking &amp;gt; 600 nm) and better temporal control of the labeling.