The Application of Fluorescent and Photosensitive Analogues of Guanine Nucleotides to the Function and Structure of G-Binding Proteins

  • J. F. Eccleston
  • T. F. Kanagasabai
  • D. P. Molloy
  • S. E. Neal
  • M. R. Webb

Abstract

In order to understand the mechanism of the interaction of a guanine nucleotide-binding protein at the molecular level it is necessary to determine the number of key intermediates of the process, to measure their rates of interconversion and ideally to gain structural information about the changes occurring during transitions between the intermediates. Our work has concentrated on defining the kinetic mechanism of elongation factor Tu (EF-Tu) on interaction with mRNA programmed ribosomes (Eccleston et al., 1985) and in the GTPase of p21N-ras (S.E. Neal, J.F. Eccleston, A. Hall, & M.R. Webb, unpublished results). These studies depended on measurements of GTP cleavage and, in the first case, peptide bond formation. However, these measurements at most give limited information about binding steps, protein isomerisations and dissociation steps which are essential features in the processes mediated by these GTPases. This information can be obtained by the use of spectroscopic probes which can be introduced into many different positions in the system.

Keywords

Thiol Adenine Epoxide Purine Photolysis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Braxton, S., and Yount, R.G., 1988, The synthesis of a novel class of ribose-modified nucleotide analogues. I. Affinity purification of skeletal myosin subfragment-1, Biophys. J., 53:178a.Google Scholar
  2. Caretta, A., Cavaggioni, A., and Sorbi, R.T., 1985, Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments, Eur. J. Biochem., 153:49–53.PubMedCrossRefGoogle Scholar
  3. Colman, R.F., 1983, Affinity labelling of purine nucleotide sites in proteins, Ann. Rev. Biochem., 52:67–91.PubMedCrossRefGoogle Scholar
  4. Cremo, C.R., and Yount, R.G., 1987, 2x′;-Deoxy-3′-O-(4-benzoylbenzoyl)- and 3′ (2′)-O-(4-benzoylbenzoyl)-1,N6-ethanoadenosine 5′-diphosphate, fluorescent photoaffinity analogues of adenosine 5′-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1, Biochemistry. 26:7524–7534.PubMedCrossRefGoogle Scholar
  5. Eccleston, J.F., 1981, Spectroscopic studies of the nucleotide binding site of elongation factor Tu from Esherichia coli. An approach to characterizing the elementary steps of the elongation cycle of protein biosynthesis, Biochemistry. 20:6265–6272.PubMedCrossRefGoogle Scholar
  6. Eccleston, J.F., 1984, A kinetic analysis of the interaction of elongation factor Tu with the guanosine nucleotides and elongation factor Ts, J. Biol. Chem., 259:12997–13003.PubMedGoogle Scholar
  7. Eccleston, J.F., and Trentham, D.R., 1977, The interaction of chromophoric nucleotides with subfragment 1 of myosin, Biochem. J., 163:15–29.PubMedGoogle Scholar
  8. Eccleston, J.F., Dix, D.B., and Thompson, R.C., 1985, The rate of cleavage of GTP on the binding of Phe-tRNA elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli, Biol. Chem.. 260:16237–16241.Google Scholar
  9. Eccleston, J.F., Gratton, E., and Jameson, D.M., 1987, Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady state and time-resolved fluorescence studies, Biochemistry, 26:3902–3907.PubMedCrossRefGoogle Scholar
  10. Eccleston, J.F., Kanagasabai, T.F., and Geeves, M.A., 1988, The kinetic mechanism of the release of the nucleotide from elongation factor Tu promoted by elongation factor Ts determined by pressure relaxation studies, J. Biol. Chem., 263:4668–4672.PubMedGoogle Scholar
  11. Faulhammer, H.G., Denninger, G., Hartl, P.J., Azhayer, A.V., Schwoerer, M., and Sprinzl, M., 1986, Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins, Biochim. Biophys. Acta. 884:182–190.PubMedCrossRefGoogle Scholar
  12. Griffin, B.E., Jarman, M., Reese, C.B., Sulston, J.E., and Trentham, D.R., 1966, Some observations relating to acyl mobility in aminoacyl soluble ribonucleic acid, Biochemistry. 5:3638–3649.CrossRefGoogle Scholar
  13. Hiratsuka, T., 1983, New ribose-modified fluorescent analogues of adenine and guanine nucleotides available as substrates for various enzymes, Biochim. Biophys. Acta. 742:496–508.PubMedCrossRefGoogle Scholar
  14. Hiratsuka, T., 1984, Distinct structures of ATP and GTP complexes in the myosin ATPase, J. Biochem. Tokyo, 96:155–162.PubMedGoogle Scholar
  15. Hobbs, J.B., and Eckstein, F., 1977, A general method for the synthesis of 2′-azido-2′-deoxy- and 2′-amino-2′-deoxyribofluranosyl purines, J. Org. Chem., 42:714–719.PubMedCrossRefGoogle Scholar
  16. Jameson, D.M., Gratton, E., and Eccleston, J.F., 1987, Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts, Biochemistry. 26:3894–3901.PubMedCrossRefGoogle Scholar
  17. Jeng, S.J., and Guillory, R.J., 1975, The use of aryl azido ATP analogues as photoaffinity labels for myosin ATPase, J. Supramol. Struct., 3:448–468.PubMedCrossRefGoogle Scholar
  18. Jurnak, F., 1985, Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins, Science. 230:32–36.PubMedCrossRefGoogle Scholar
  19. Jurnak, F., 1988, The three-dimensional structure of c-H-ras p21: implications for oncogene and G protein studies, Trends in Biochem. Sci., 13:195–198.CrossRefGoogle Scholar
  20. Kapuler, A.M., and Reich, E., 1971, Some stereochemical requirements of Esherichia coli ribonucleic acid polymerase. Interaction with conformationally restricted ribonucleoside 5′-triphosphates: 8-bromoguanosine, 8-ketoguanosine, and 6-methylcytidine triphosphates, Biochemistry. 10:4050–4061.PubMedCrossRefGoogle Scholar
  21. Kulikowska, E., Bzouska, A., Wierzchowski, J., and Shugar, D., 1986, Properties of two unusual, and fluorescent, substrates of purine-nucleoside Phosphorylase: 7-methyIguanosine and 7-methylinosine, Biochim. Biophys. Acta. 874:355–363.PubMedCrossRefGoogle Scholar
  22. Kusmierek, J.T., Jensen, D.E., Spengler, S.J., Stolarski, R., and Singer, B., 1987, Synthesis and properties of N2, 3-ethenoguanosine and N2, 3-ethenoguanosine 5′-diphosphate, J. Org. Chem., 52:2374–2378.CrossRefGoogle Scholar
  23. LaCour, T.F.M., Nyborg, J., Thirup, S., and Clark, B.F.C., 1985, Structural details of the binding of guanosine diphosphate to elongation factor Tu from Esherichia coli as studied by X-ray crystallography, EMBO J., 4:2385–2388.Google Scholar
  24. Leonard, N.J., and Tolman, G.L., 1975, Fluorescent nucleosides and nucleotides, Ann. NY Acad. Sci., 255:43–58.PubMedCrossRefGoogle Scholar
  25. Leonard, N.J., 1984, Etheno-substituted nucleotides and coenzymes: fluorescence and biological activity, CRC Crit. Rev. Biochem., 15:125–199.PubMedCrossRefGoogle Scholar
  26. Miller, D., and Weissbach, H., 1977, Factors involved in the transfer of aminoacyl-tRNA to the ribosome, in “Molecular mechanisms of protein biosynthesis,” H. Weissbach and S. Pestka, eds., Academic Press, London.Google Scholar
  27. Nakanishi, T., Tomita, F., and Suzuki, T., 1974, Production of a new aminonucleoside “9-(2′-amino-2′-deoxypentofuranosyl) guanine” by Aerobacter-sp, Agri. Biol. Chem., 38: 2465–2469.CrossRefGoogle Scholar
  28. Potter, R.L., and Haley, B.E., 1983, Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogues: techniques and applications, Methods Enzymol., 91:613–633.PubMedCrossRefGoogle Scholar
  29. Prendergast, F.G., Meyer, M., Carlson, G.C., Iida, S., and Potter, J.D., 1983, Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonapthalene (Acrylodan). A thiol-selective, polarity-sensitive fluorescent probe, J. Biol. Chem., 258:7541–7544.PubMedGoogle Scholar
  30. Stryer, L., 1978, Fluorescence energy transfer as a spectroscopic ruler, Ann. Rev. Biochem. 47:819–846.PubMedCrossRefGoogle Scholar
  31. Ward, P.C., Reich, E., and Stryer, L., 1969, Fluorescence studies of nucleotides and polynucleotides, J. Biol. Chem. 244:1228–1237.PubMedGoogle Scholar
  32. Weber, G., and Farris, F.J., 1979, Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino) mapthalene, Biochemistry. 18:3075–3078PubMedCrossRefGoogle Scholar
  33. Weber, I.T., Steitz, T.A., Bubis, J., and Taylor, S.S., 1987, Predicted structures of cAMP binding domains of Type I and II regulatory subunits of cAMP-dependent protein kinase, Biochemistry. 26:343–351.PubMedCrossRefGoogle Scholar
  34. Weigand, G., and Kaleja, R., 1976, Fluorescent guanosine-nucleotide analogues suitable for photoaffinity-labelling experiments, Eur. J. Biochem., 65:473–479.CrossRefGoogle Scholar
  35. Wittinghofer, A., Warren, W.F., and Leberman, R., 1977, Structural requirements of the GDP binding site of elongation factor Tu, FEBS Lett., 75:241–243.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • J. F. Eccleston
    • 1
  • T. F. Kanagasabai
    • 1
  • D. P. Molloy
    • 1
  • S. E. Neal
    • 1
  • M. R. Webb
    • 1
  1. 1.National Institute for Medical ResearchLondonUK

Personalised recommendations