Student design and characterization of visible DHFR fusions for biochemistry tools to improve learning during lab exercises.

Student feedback from an undergraduate biochemistry lab course suggested the use of visibly traceable proteins may assist learning. Based on this feedback, we used guided inquiry lab exercises where students developed and characterized a suite of fluorescent protein-dihydrofolate reductase (DHFR) fusions as tools for a biochemistry teaching lab. In contrast to the unfused versions, members of this suite are well-expressed, soluble, visible, highly stable, and easily characterized. The color of mCherry and EGFP fluorescent fusions with microbial DHFR allows students to visibly track their target protein from expression through purification under ambient light, while fusions with BFP are visible under UV-light. Fusions were made to both wild-type and kinetically enhanced DHFR variants. Importantly, we found that fluorescent protein fusions with DHFR did not kinetically interfere as the KM and kcat values were not remarkably altered from the unfused variant. With these fusions, students can easily measure kinetic parameters under steady-state conditions with readily available substrate and common laboratory spectrophotometers. Additionally, students also determined IC50 values of trimethoprim for DHFR. These exercises can be completed in a series of up to six lab periods and we have included the protocols for instructors who wish undertake a similar series of experiments in their biochemistry teaching labs. Using these visible fusion enzymes with subsequent students, we observed potential learning gains on a course assessment and received positive student feedback. We suggest that the often over-looked element of visual cues in a biochemistry lab may be an exploitable component of learning.

Otimização de novos inibidores da di-idrofolato redutase de Mycobaterium tuberculosis (mtDHFR): Docking molecular, sí­ntese, avaliação da inibição enzimática e da atividade antimicobacte / Optimization of new dihydrofolate reductase inhibitors of mycobacterium tuberculosis (mtdhfr): molecular docking, synthesis, evaluation of enzyme inhibition and antimycobacterial activity

A tuberculose (TB) é considerada uma das principais doenças infecciosas e apresenta fatores críticos como a relação com o HIV/AIDS, tratamento longo e a resistência a múltiplos fármacos. A enzima di-hidrofolato redutase das micobactérias (mtDHFR) é um alvo pouco explorado e apresenta grande potencial para o desenvolvimento de novos fármacos contra TB. Estudos preliminares obtiveram fragmentos com baixa afinidade à mtDHFR, entretanto com potencial para otimização. Com isso, o fragmento foi usado como protótipo para a proposição de 22 análogos. Os compostos foram planejados utilizando informações sobre ligantes e a estrutura tridimensional de mtDHFR, além do biososterismo como estratégia norteadora. Os ensaios de docking molecular com a mtDHFR revelaram que os análogos propostos tiveram escores interessantes e, além disso, a inserção de substituintes demonstrou favorecer a ligação à enzima, o que corroborou o planejamento. Com isso, sintetizou-se 22 análogos planejados e o protótipo MB872, por meio de protocolos de alquilação, hidrólise e cicloadição 1,3 dipolar para os compostos com anéis triazol e tetrazol. Os compostos foram obtidos com rendimentos de bom a ótimo (60 ~ 90%) e suas estruturas foram elucidadas por RMN 1H e 13C. Os resultados do ensaio de inibição enzimática corroboraram com os dados de docking, uma vez que a presença do grupo carboxílico revelou ser importante para a atividade. Além disso, alguns dos compostos revelaram atividades interessantes, entre 8 a 40 µM, sendo que o mais ativo apresentou IC50 de 7 µM. Ensaios de cinética enzimática com o análogo mais ativo indicou uma inibição não competitiva com o substrato natural da enzima, uma vez que os valores de Km se mantiveram constantes, enquanto Vmax decaiu (0,22 µM e 0,43 - 0,34 ΔFU/min, respectivamente). Os análogos sintetizados foram mandados para ensaio in vitro para avaliar a atividade frente a micobactéria

Tuberculosis (TB) is an important infectious disease and presents critical factors such as the relationship with HIV / AIDS, long treatment and resistance to multiple drugs. The enzyme dihydrofolate reductase from mycobacteria (mtDHFR) is a poorly explored and presents great potential to be a target for new drugs against TB. Preliminary studies have obtained fragments with low affinity to mtDHFR, but with potential to become lead compounds. Therefore, the fragment was used as a prototype for 22 analogues proposed in this work. The compounds were designed using bioisosterism, information about ligands and the three-dimensional structure of mtDHFR. Molecular docking assays with mtDHFR revealed satisfactory scores for anlogues. Furthermore, the insertion of substituents seemed to increase the affinity with the enzyme. Thereby, twenty two analogues and prototype were synthesized using alkylation, hydrolysiss and 1,3-dipolar cycloaddition methods. The compounds were obtained in good yields (60 ~ 90%) and their structures were elucidated with 1H and 13C NMR spectroscopy. The enzymatic affinity assay corroborates docking results, because the presence of carboxyl group showed to be important for the activity. Furthermore, some of the compounds revealead interesting activities, ranging 8 to 40 µM. The most active showed IC50 of 7 µM and enzyme kinetics assays indicated noncompetitive inhibition with natural enzyme substrate. The synthesized analogs were sent for in vitro assay to assess mycobacteria activity

Selective purification and chemical labeling of a target protein on ruthenium photocatalyst-functionalized affinity beads.

Selective purification and chemical labeling of a target protein in a protein mixture were simultaneously achieved on the surface of affinity beads functionalized with ligands, such as benzenesulfonamide and methotrexate (MTX), and a ruthenium complex containing 2,2'-bipyridine-4,4'-dicarboxylic acid (dcbpy). Chemical labeling of the target protein with a tyrosine radical trapper (TRT) proceeded on the surface of the beads when the target protein was in close proximity to the ruthenium photocatalyst. Both the protein purification and chemical labeling abilities of the affinity beads functionalized with ruthenium photocatalyst were not compromised after recycling several times. Dihydrofolate reductase (DHFR) endogenously expressed in HeLa cells was detected by chemical labeling with biotin-TRT on the affinity beads with high sensitivity compared to the conventional silver staining method.

Single-Molecule Analyte Recognition with ClyA Nanopores Equipped with Internal Protein Adaptors.

Nanopores have been used to detect molecules, to sequence DNA, or to investigate chemical reactions at the single-molecule level. Because they approach the absolute limit of sensor miniaturization, nanopores are amenable to parallelization and could be used in single-cell measurements. Here we show that single enzymes can be functionally and reversibly trapped inside the confined space of a ClyA nanopore. Remarkably, the binding of ligands to the internalized proteins is mirrored by specific changes to the nanopore conductance. Conveniently, the manipulation of the charge of the protein allowed increasing of the residence time of the protein inside the nanopore. Nanopores with internalized protein adaptors can be used to study proteins in real time or can be incorporated into inexpensive portable devices for the detection of analytes with high selectivity.

Association between expression of thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase and efficacy of pemetrexed in advanced non-small cell lung cancer.

BACKGROUND: Pemetrexed inhibits three key folate enzymes: thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The relationship between the clinical efficacy of pemetrexed and the expression of folate enzymes in lung cancer cells is unknown. The purpose of this study was to determine whether TYMS, DHFR, and GARFT expression affect the therapeutic efficacy of pemetrexed. PATIENTS AND METHODS: Participants (n=50) were patients with advanced non-small cell lung cancer (NSCLC) treated with pemetrexed. Samples were obtained by tumor biopsy before treatment. We isolated cancer cells from formalin-fixed paraffin-embedded tissues using laser microdissection, and mRNA levels were analyzed using real-time reverse transcription polymerase chain reaction. Protein expression was evaluated using immunohistochemistry. We assessed the association between TYMS, DHFR, and GARFT expression and the therapeutic efficacy of pemetrexed. RESULTS: The median age was 66.8 years. Compared to healthy tissues, the relative TYMS mRNA expression ranged from 0.001 to 41.613 (mean 4.638 ± 1.357), and was significantly lower in responders compared to non-responders (1.671 ± 0.844 versus 5.978 ± 1.895, p=0.0142). Progression-free survival was prolonged in patients with lower TYMS mRNA expression compared to those with higher TYMS mRNA expression, but the difference was not statistically significant (18.0 versus 13.3 weeks, p=0.3001). DHFR and GARFT mRNA expression did not correlate with the efficacy of pemetrexed. CONCLUSION: We specifically analyzed TYMS, DHFR, and GARFT mRNA expression levels in lung cancer cells from biopsy specimens using laser microdissection. TYMS mRNA expression affected the therapeutic efficacy of pemetrexed and could therefore constitute a useful predictive biomarker for NSCLC patients receiving pemetrexed.

Second-generation covalent TMP-tag for live cell imaging.

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag.

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.

Impairment of gastric nitrergic and NRF2 system in apolipoprotein E knockout mice.

BACKGROUND AND AIM: Gastric motility dysfunction is most commonly seen in diabetic and idiopathic gastroparesis patients. Recently we reported that impaired nitrergic relaxation and a reduced NO (nitric oxide) bioavailability were responsible for gastric motility dysfunction in diabetic female rats. One of the main factors involved in the inactivation of the nitrergic system is oxidative stress commonly seen in diabetic patients. Hyperlipidemia may also be one of the detrimental causes for impaired gastric motility associated with diabetes. In the current study, we investigated whether apolipoprotein E knockout mice (ApoE-KO), an oxidative stress animal model with a hyperlipidemia burden, also displays an impaired nitrergic system. To test this, nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric pyloric strips prepared from C57BL WT or ApoE-KO female mice. Protein expression was determined by Western blots. RESULTS: Nitrergic relaxation was reduced in gastric strips from ApoE-KO versus WT mice. Protein levels of nNOS (neuronal nitric oxide synthase), GCH-1 (GTP cyclohydrolase 1), Nrf2 (nuclear factor E-2 related factor 2) and GCSc (glutamate-cysteine ligase catalytic) were also reduced in ApoE-KO compared to controls, with no significant change in GCSm (glutamate-cysteine ligase modifier) and HO-1 (heme oxygenase 1). The activities of DHFR (dihydrofolate reductase) and antioxidant enzymes were also reduced in ApoE-KO mice. CONCLUSIONS: This novel study is the first to reveal that a deficiency in ApoE impairs gastric motility functions, and that hyperlipidemia and the suppression of selective antioxidants may be an underlying mechanism for this pathological change.

Protein-fragment complementation assays for large-scale analysis, functional dissection and dynamic studies of protein-protein interactions in living cells.

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Assays can be performed in any cell type or model organism that can be transformed or transfected with gene expression DNA constructs. Here we focus on recent applications of PCA in the budding yeast, Saccharomyces cerevisiae, that cover the gamut of applications one could envision for studying any aspect of PPIs. We present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR), reporter PCA, and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present methods to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs.

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays.

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.

Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer.

BACKGROUND: Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR) in colorectal cancer, and to evaluate its prognostic significance following adjuvant chemotherapy, since these enzymes are closely related to efficacy of 5-fluorouracil (5FU). PATIENTS AND METHODS: Consecutive patients (n = 314), who were completely resected for colorectal cancer stages II-IV and adjuvantly treated with 5-FU were retrospectively evaluated. Paraffin embedded tumor specimens were assessed for gene copies per nucleus of TYMS, TP and DHFR by fluorescence in situ hybridisation (FISH) using specific peptide nucleic acid probes. Outcome according to gene copies per nucleus above and below the median were compared. Also TYMS expression, assessed by immunohistochemistry, was associated with TYMS copies per nucleus. RESULTS: The number of gene copies per nucleus were 1.7 (0.7-2.8), 1.8 (0.9-3.1) and 1.8 (1.1-2.7) median (range) for TYMS, TP and DHFR, respectively. TYMS expression was directly associated with TYMS genes per nucleus (p = 0.05). Cox multivariate analysis, adjusted for the prognostic impact of disease stage, vascular tumor invasion, and bowel obstruction at resection, revealed that high TYMS gene copy number was associated with significantly higher risk of recurrence (HR = 1.6; 95%CI 1.1-2.2; p = 0.02) and death (HR = 1.6; 95%CI 1.1-2.3; p = 0.01). No significant differences in outcome appeared according to TP and DHFR gene ratios. CONCLUSION: Aberration of TYMS gene is of significance to expression of TYMS, which may influence the biology and 5-FU sensitivity of colorectal cancer. This may be utilized in the allocation of patients for treatment approaches and for decision on follow-up programs.

Role of angiotensin II on dihydrofolate reductase, GTP-cyclohydrolase 1 and nitric oxide synthase expressions in renal ischemia-reperfusion.

BACKGROUND: The present study was conducted to investigate the role of renal ischemia-reperfusion (IR) and angiotensin II (ANG II) on mRNA and protein levels of renal dihydrofolate reductase (DHFR), GTP-cyclohydrolase 1 (GTP- CH 1), and endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively). METHODS: Male Wistar rats were sham operated or received IR (30 min occlusion, and reperfusion for 1 day). Each group was treated separately with water, angiotensin-converting enzyme inhibitor (ACEI) and ANG II receptor type 1 blocker (ARB) for 1 day before the sham operation or IR, and continuously for 1 day after the operation. The mRNA and protein levels were detected by RT-PCR and Western blot, respectively. RESULTS: IR decreased DHFR mRNA and protein levels (p < 0.01), both of which were restored by ACEI or ARB, whereas GTP-CH 1 expression was unaltered. IR suppressed eNOS dimer while enhancing the monomer (p < 0.01). IR augmented iNOS mRNA, total iNOS protein and iNOS monomer (all p < 0.01) which were attenuated by ACEI or ARB. CONCLUSION: Our study is the first to demonstrate that the heightened ANG II in IR, via stimulation of ANG II receptor type 1, suppresses DHFR and eNOS dimer, while activating both iNOS mRNA and protein levels.

Development of a chemical screening system using aqueorin-fused protein.

We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.

Proteomic analysis for the identification of proteins related to methotrexate resistance.

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 micromol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.

Histochemical study of the skin affected by certain autoimmune diseases.

This study puts forth the morphological and metabolic modifications that occurred in the skin affected by Pemphigus vulgaris, modifications that have been studied by histoenzymatic techniques. There were studied skin biopsies from patients suffering from Pemphigus vulgaris, hospitalized in Dermatology services in Bucharest. The pieces were sectioned at cryotome, at 3 degrees C and then they were studied with histochemical methods, in order to evidentiate the activity of the following enzymes: NADH(2)-cytochrome-c-reductase, lactate-dehydrogenase (LDH), dihydrofolic-reductase (DHFR), folic acid (FA), ATP-ase pH 9.4, leucin-amino-peptidase (LAP). The enzymatic activity was appreciated in all skin components: epidermal layers, connective tissue cells and inflammatory cells from the superficial and deep dermis, blood vessels, nerve fibers and encapsulated corpuscles. In the affected skin, there were observed modifications of all enzymes studied. The study revealed the following aspects: the balance between oxybiotic and anoxybiotic metabolism in the epidermal cells changes from the predominance of the former in the healthy skin, to the dominance of the latter in the affected skin, an active turn-over of the nucleic acids and increased synthesis of amino acids in the dermal cell population (mainly in the fibroblasts and mast cells), morphological and functional alteration of mitochondria, lysosomes and cellular membranes.

Modulation of specific protein expression levels by PTEN: identification of AKAP121, DHFR, G3BP, Rap1, and RCC1 as potential targets of PTEN.

The tumor suppressor PTEN is mutated in a high percentage of human cancers, and is implicated in pathways regulating cell growth, proliferation, survival, and migration. Despite significant advances, our understanding of its mechanisms of action remains incomplete. We have used a high-throughput proteomic immunoblotting approach to identify proteins whose expression levels are modulated by PTEN. Out of over 800 proteins screened, 22 proteins showed significant changes in expression. Five proteins that exhibited two-fold or greater changes in expression level were further characterized. AKAP121 and G3BP expression was reduced, while dihydrofolate reductase (DHFR), Rap1 and RCC1 expression was elevated in response to PTEN expression in a PTEN-null T-cell leukemia line. The phosphatase activity of PTEN was required for these effects. However, direct inhibition of PI-3 Kinase could mimic PTEN in modulating expression of DHFR, G3BP, Rap1 and RCC1, but not AKAP121. Real-time PCR showed that the effects of PTEN were primarily post-transcriptional, and would not have been revealed by mRNA-based screens. We conclude from these data that PTEN can modulate the expression level of a number of different proteins. The identified proteins have the potential to serve as previously unrecognized effectors of PTEN, and suggest the existence of additional complexity in the modes by which PTEN can regulate cellular biology.

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus.

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

Retroviral transduction of a mutant dihydrofolate reductase-thymidylate synthase fusion gene into murine marrow cells confers resistance to both methotrexate and 5-fluorouracil.

Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene. The DHFR F/S-TS G52S fusion protein is functional and exhibits kinetic properties similar to that of the individual mutant enzymes. NIH 3T3 cells and mouse bone marrow progenitors retrovirally transduced with the fusion DHFR F/S-TS G52S cDNA provided similar levels of resistance to MTX and 5-FU as cells expressing the individual mutant enzymes and higher levels of resistance to MTX than cells expressing DHFR F/S from the 3' end of a bicistronic gene. As MTX and 5-FU are used in combination therapy for diseases such as breast and colon cancer, this fusion gene may be useful in the clinic to reduce myelosuppressive toxicity associated with this drug combination.

A fluorogenic dye activated by the staudinger ligation.

Specific labeling of biomolecules with biochemical and biophysical probes is a central element of proteomics research. Here we describe a coumarin-phosphine dye that undergoes activation of coumarin fluorescence upon Staudinger ligation with azides. Since azides can be metabolically incorporated into cellular proteins and oligosaccharides, this dye may be a useful tool for profiling proteins and their posttranslational modifications.