Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe³⁺-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.     

Tyrosine specific sequential labeling of proteins

We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an alkyne or azide function, respectively, and (c) the selective modification human serum albumin by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of tyrosine residues with an alkyne bearing linker and a subsequent azide–alkyne click reaction with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed modification of the tyrosines may also be used to introduce other kinds of tags. With these reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly or in a sequential manner.     

Intracellular binding of ethidium studied by photoaffinity labeling in vivo

The azide analog of ¹⁴C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles. The ¹⁴C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions.     

Behavior of a photoactivatable analog of cholesterol, 6-photocholesterol, in model membranes

6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42–49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725–1736], and to probe lipid–protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143–154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.     

Photoaffinity labeling of acetylcholine receptor in millisecond time scale

Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein. In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca2+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.     

Interaction of N-(2-Nitro-4-Azidophenyl)-Serotonin with Two Types of Monoamine Oxidase in Rat Brain

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.     

Strain and strain rate activation of G proteins in human endothelial cells

The endothelium is known to sense and respond to its physical environment, but the underlying mechanisms and early events of endothelial cell mechanotransduction are not well understood. The present study measured G protein activation by mechanical strain in human umbilical vein endothelial cells (HUVEC) directly by photoincorporation of a hydrolysis resistant, radiolabeled GTP analog. Ten percent uniaxial strain at a strain rate of 20% s(-1) over 1min activated a 38kDa Galpha subunit 167+/-17% relative to controls, while 2% cyclic strain failed to significantly activate the protein (117+/-19%). A single cycle of 10% strain at 20% s(-1) strain rate activated the Galpha subunit 152+/-25%, while activation at the same strain but lower strain rate (0.3% s(-1)) was not significantly different from controls (116+/-12%). Western blot analysis identified the 38kDa protein as Galpha(q/11). These results demonstrate the rapid activation of G proteins in HUVEC by cyclic uniaxial strain in a strain- and strain rate-dependent manner.     

Photoaffinity labeling of platelet thrombin-binding proteins

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins; platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequences of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or α₂-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.     

Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors

The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 °C to 4 °C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors.     

Saturation fluorescence labeling of proteins for proteomic analyses

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations, including the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP), pH, incubation time, linearity of labeling, and saturating dye/protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific versus nonspecific labeling in the presence of thiourea are also discussed. We found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, and α-lactalbumin) is achieved at 50- to 100-fold excess of the uncharged maleimide-functionalized BODIPY dyes over Cys. We confirmed our previous findings, and those of others, that the maleimide dyes are not affected by the presence of 2 M thiourea. Moreover, we established that 2 mM TCEP used as reductant is optimal. We also established that labeling is optimal at pH 7.5 and complete after 30 min. Low nonspecific labeling was gauged by the inclusion of non-Cys-containing proteins (horse myoglobin and bovine carbonic anhydrase) to the labeling mixture. We also showed that the dye exhibits little to no effect on the two-dimensional mobilities of labeled proteins derived from cells.     

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.     

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent D-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K(i) values of 22+/-5 microM for TIPDG and 12+/-2 microM for BzG. These K(i) values are comparable to those of their parent compounds arbutin (25+/-6 microM) and phlorizin (8+/-1 microM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure-function relationship of SGLT1 and other SGLT1 family transporter proteins.     

Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes

Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue ¹²⁵I-labeled azidobenzoyl (Trp¹¹)-neurotensin or by cross-linking (monoiodo-Tyr³)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different.     

Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).     

Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans

To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([alpha-(32)P]8-azidoATP; IC(50)=0.76 microM) and drug-substrates ([(3)H]azidopine and [(125)I]iodoarylazidoprazosin; IC(50) approximately 12 microM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 microM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy.     

Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent label molecule. To minimize the amount of additional sample handling and time associated with such labeling procedures, not to mention the sometimes-stringent pH and temperature controls they require, noncovalent labeling is presented as a viable alternative. This review article considers established methods for noncovalent labeling of proteins for their subsequent analysis by CE-LIF. Label molecules suitable for excitation and emission in the ultraviolet, visible, and near-infrared regions of the spectrum are enumerated for a variety of protein analytes.     

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [¹³C₆]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from ¹³C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.     

Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol