guanosine-triphosphate and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Improving the yield of unnatural amino acid incorporation is an important challenge in producing novel designer proteins with unique chemical properties. Here we examine the mechanisms that restrict the incorporation of the fluorescent unnatural amino acid εNH2-Bodipy576/589-lysine (BOP-Lys) into a model protein. While the delivery of BOP-Lys-tRNA(Lys) to the ribosome is limited by its poor binding to elongation factor Tu (EF-Tu), the yield of incorporation into peptide is additionally controlled at the step of BOP-Lys-tRNA release from EF-Tu into the ribosome. The unnatural amino acid appears to disrupt the interactions that balance the strength of tRNA binding to EF-Tu-GTP with the velocity of tRNA dissociation from EF-Tu-GDP on the ribosome, which ensure uniform incorporation of standard amino acids. Circumventing this potential quality control checkpoint that specifically prevents incorporation of unnatural amino acids into proteins may provide a new strategy to increase yields of unnatural polymers.

Topics: Bacterial Proteins; Boron Compounds; Escherichia coli; Fluorescent Dyes; Guanosine Triphosphate; Kinetics; Lysine; Models, Molecular; Peptide Elongation Factor Tu; Ribosomes; RNA, Transfer; Thermus thermophilus

Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP.

Topics: Bacillus subtilis; Bacterial Proteins; Boron Compounds; Calorimetry; Fluorescent Dyes; Guanosine Triphosphate; Light; Models, Molecular; Photoreceptors, Microbial; Spectrometry, Fluorescence

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.

Topics: Boron Compounds; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Electrophoresis, Capillary; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Kinetics; Microfluidic Analytical Techniques; RGS Proteins; Rhodamines

Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max cat) steady-state and K(m) values were determined from assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Galpha and Galpha(i1) were 8.3 +/- 1 x 10(-3) and 3.0 +/- 0.2 x 10(-2) s(-1), respectively, and K(m) values were 120 +/- 60 and 940 +/- 160 nm. Assays with BGTPgammaS yielded maximum turnover numbers of 1.6 +/- 0.1 x 10(-4) and 5.5 +/- 0.3 x 10(-4) s(-1) for Galpha and Galpha(i1); K(m) values were 14 (o)(+/-)8 and 87 +/- 22 nm. Acceleration of Galpha GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY(R) FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 mum YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Galpha subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPgammaS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.

Topics: Biochemistry; Boron Compounds; Buffers; Calibration; Electrophoresis, Capillary; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Triphosphate; Hydrolysis; Kinetics; Models, Chemical; Mutation; Spectrometry, Fluorescence; Time Factors

Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5'-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5'-diphosphate with high intrinsic exchange rates in the presence of Mg(2+) ions, thereby acting as synthetic, low molecular weight guanine nucleotide exchange factors. The accompanying large fluorescence enhancements (as high as 12-fold), caused by a reduction in guanine quenching of the environmentally sensitive BODIPY dye fluorescence on protein binding, allow for real-time monitoring of this spontaneous nucleotide exchange in the visible spectrum with high signal-to-noise ratios. Binding affinities increased with longer aliphatic linkers connecting the nucleotide and BODIPY fluorophore and were in the 10-100 nM range. Steady-state and time-resolved fluorescence spectroscopy showed an inverse relationship between linker length and fluorescence enhancement factors and differences in protein-bound fluorophore mobilities, providing optimization criteria for future applications of such compounds as efficient elicitors and reporters of nucleotide exchange. EDTA markedly enhanced nucleotide exchange, enabling rapid loading of GTPases with these probes. Differences in active site geometries, in the absence of Mg(2+), caused qualitatively different reporting of the bound state by the different analogs. The BODIPY analogs also prevented the interaction of Cdc42 with p21 activated kinase. Together, these results validate the use of these analogs as valuable tools for studying GTPase functions and for developing potent synthetic nucleotide exchange factors for this important class of signaling molecules.

Topics: Boron Compounds; Edetic Acid; Fluorescent Dyes; GTP Phosphohydrolases; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Molecular Weight; Spectrometry, Fluorescence

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.

Topics: Adenosine Triphosphate; Algorithms; Boron Compounds; Cell Membrane; Crystallography, X-Ray; Dose-Response Relationship, Drug; Electrons; Guanine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Hydrolysis; Kinetics; Ligands; Models, Chemical; Models, Molecular; Phosphates; Protein Binding; Protein Conformation; Spectrometry, Fluorescence

Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.

Topics: Animals; Boron Compounds; Fluorescence; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Humans; Protein Subunits; RGS Proteins; Signal Transduction; Spectrometry, Fluorescence

Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding.

Topics: Boron Compounds; Escherichia coli; Fluorescent Dyes; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Protein Binding; RGS Proteins

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.

Topics: Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Boron Compounds; Brain; Cromakalim; Decanoic Acids; Diazoxide; Fluorescent Dyes; Glyburide; Guanosine Triphosphate; Hydroxy Acids; Kinetics; Ligands; Liver; Membrane Proteins; Mitochondria; Mitochondria, Liver; Models, Biological; Myocardium; Oxygen Consumption; Potassium Channels; Proteolipids; Rats; Time Factors; Vasodilator Agents

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.

Topics: Actins; ADP-Ribosylation Factors; Animals; Anti-Bacterial Agents; Boron Compounds; Brefeldin A; Cell Line; Ceramides; Coatomer Protein; Cyclopentanes; Cytoskeleton; Erythrocytes; Fluorescent Dyes; Golgi Apparatus; GTP-Binding Proteins; Guanosine Triphosphate; Macrolides; Macrophages; Mice; Microscopy, Fluorescence; Microtubule-Associated Proteins; Receptors, IgG; Transfection