Fluorescent nucleotide analogs: synthesis and applications.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J F Eccleston
Publications and source records attributed to J F Eccleston.
Characterisation of the interaction of normal and mutant Rho-GAP with Rho family proteins.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.
The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.
Animals↗
Mechanism of inhibition by arachidonic acid of the catalytic activity of Ras GTPase-activating proteins.
Ras is a guanine nucleotide-binding protein that acts as a molecular switch controlling cell growth. The Ras GTPase-activating proteins (GAPs) p120-GAP and neurofibromin are candidates as Ras effectors. The GTPase-activating activity of both proteins is inhibited by mitogenic lipids, such as arachidonic acid and phosphatidic acid, and differential inhibition of the two GAPs led to the hypothesis that both were effectors in a Ras-controlled mitogenic pathway (Bollag, G., and McCormick, F. (1991) Nature 351, 576-579). We have studied the mechanism of inhibition by arachidonic acid in three ways: first, by measurements of catalytic activity under multiple turnover conditions; second, using p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid as a fluorescent probe for ligands binding to GAPs; and third, by using a scintillation proximity assay to measure direct binding of Ras to neurofibromin. We found no significant differential inhibition between p120-GAP and neurofibromin by arachidonic acid. The inhibition by arachidonic acid included a major component that is competitive with Ras GTP. These data suggest that insomuch as the mitogenic effects of lipids are mediated via inhibition of GAPs, GAPs are not Ras effector proteins. Additionally, lipids can exert a non-competitive type effect, consistent with a protein denaturing activity, making difficult extrapolations from in vitro data to the situation within cells, and possibly explaining the variability of literature data on inhibition by lipids.
Arachidonic Acid↗
Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.
The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Energy Transfer↗
Kinetics of interaction between normal and proline 12 Ras and the GTPase-activating proteins, p120-GAP and neurofibromin. The significance of the intrinsic GTPase rate in determining the transforming ability of ras.
Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.
Cell Transformation, Neoplastic↗
Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy.
An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured. EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes. The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.
Aurodox↗
Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.
The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5 degrees C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30 degrees C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12]p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5 degrees C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21,000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore.(ABSTRACT TRUNCATED AT 250 WORDS)
Chemical Phenomena↗
Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue.
The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP). Since the concentration of active p21 is important in these measurements, various assays for both total protein and active p21 were investigated. All assays gave good agreement except the filter binding assay of [3H]-GDP bound to p21, which gave values of 35-40% compared to the other methods. Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21-mant-nucleotide complexes. The rate constants of the elementary steps of the p21 intrinsic GTPase activity and the GAP activated activity were similar between GTP and mantGTP. Incubation of a stoichiometric complex of p21.mantGTP results in a biphasic decrease in fluorescence. The second phase occurs with the same rate constant as the cleavage step and is accelerated by GAP. No other steps of the mechanism are affected by GAP. Incubation of a stoichiometric complex of p21.mantGpp[NH]p also results in a biphasic decrease in fluorescence even though cleavage does not occur. This is interpreted that the cleavage step of p21.GTP is preceded by and controlled by an isomerization of the p21.GTP complex. GAP accelerates the rate constant of the second fluorescence phase occurring with p21.mantGpp[NH]p. This result shows that GAP accelerates the proposed isomerization which limits GTP cleavage rather than the cleavage step itself.
Chromatography, Gel↗
The kinetic mechanism of the GAP-activated GTPase of p21 ras.
Guanine nucleotides modified by acetylation of the ribose moiety with the small fluorophore N-methylanthranilic acid(mant) have been shown to bind to p21 ras with similar equilibrium and kinetic rate constants as the parent nucleotides. Hydrolysis of p21.mantGTP to p21.mantGDP results in a 10% decrease in fluorescence intensity occurring at the same rate as the cleavage step. A similar process occurs with the non-hydrolysable analogue mantGMP.PNP, and this has led to the proposal that a conformational change of p21.mantGTP precedes and controls the rate of the cleavage step. The fluorescence change with p21.mantGMP.PNP is accelerated in the presence of the C-terminal catalytic domain of GAP, which is consistent with this mechanism. The same conformational change does not occur with oncogenic mutants of p21 ras, Asp-12 and Val-12, but does occur with the weakly oncogenic Pro-12 mutant. Stopped flow measurements of the interaction of GAP with p21.mantGTP show an exponential decrease in fluorescence, the rate of which does not vary linearly with GAP concentration. These data imply a rapidly reversible formation of the p21.mantGTP complex with GAP followed by the isomerization of this complex. This is at least 10(5)-fold faster than the same process in the absence of GAP.
GTP Phosphohydrolases↗
Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes.
We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto-S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Good, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.
Actomyosin↗
Fluorescence approaches to the study of the p21ras GTPase mechanism.
The use of ribose-modified guanine nucleotides and tryptophan mutants of p21ras, neither of which have significant effect on the kinetic mechanism of the p21ras GTPase and the GAP-activated p21ras GTPase, will now allow a detailed kinetic study of how GAP and other regulatory proteins interact with p21ras. This will lead to a better understanding of how the relative concentrations of 'active' p21ras. GTP and 'inactive' p21ras. GDP are regulated in the cell.
GTP Phosphohydrolases↗
Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant.
Binding Sites↗
Photoreceptor channel activation by nucleotide derivatives.
Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism.
The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.
Escherichia coli↗
The kinetic mechanism of the release of nucleotide from elongation factor Tu promoted by elongation factor Ts determined by pressure relaxation studies.
The release of a chromophoric analogue of GDP, 2-amino-6-mercaptopurine riboside 5'-diphosphate (thioGDP), from its complex with elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). The mechanism of this reaction includes a ternary complex; EF-Tu.thioGDP.EF-Ts (Eccleston, J. F. (1984) J. Biol. Chem. 259, 12997-13003). This mechanism has been further investigated using pressure relaxation techniques combined with spectrophotometric measurements. The equilibrium of a solution of EF-Tu, EF-Ts, and thioGDP over a range of concentrations is perturbed on increasing the pressure to 150 atm. Rapid decrease of the pressure back to 1 atm results in a biphasic relaxation process, an initial fast phase which is complete within 1 ms followed by a slower phase. This is interpreted as the result of an isomerization of the EF-Tu.thioGDP.EF-Ts ternary complex which occurs before the release of thioGDP. Such an isomerization process may be a general feature in the release of GDP from guanosine nucleotide-binding proteins.
Guanosine Diphosphate↗
Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts.
The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.
Fluorescence↗
Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady-state and time-resolved fluorescence studies.
A fluorescent derivative of GDP was prepared by the reaction of 2'-amino-2'-deoxy-GDP with fluorescamine. This derivative binds tightly (KD approximately 4.5 X 10(-8) M) to elongation factor Tu (EF-Tu) from Escherichia coli. The emission properties, including spectra, polarizations, and lifetimes, for fluorescamine-GDP free in solution and bound to EF-Tu are presented. Emission data on the fluorescamine-ethylamine conjugate are also given. A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2-80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively. Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe. Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values. The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10(-3) S-1 was determined. These results demonstrate the utility of this 2'-amino-2'-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems.
Absorption↗