Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1987 Jun;51(6):865–873. doi: 10.1016/S0006-3495(87)83414-8

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study.

L X Chen, J W Longworth, G R Fleming
PMCID: PMC1330020  PMID: 3038204

Abstract

The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A.

Full text

PDF
866

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arata Y., Kimura S., Matsuo H., Narita K. Proton and phosphorus nuclear magnetic resonance studies of ribonuclease T1. Biochemistry. 1979 Jan 9;18(1):18–24. doi: 10.1021/bi00568a003. [DOI] [PubMed] [Google Scholar]
  2. Beechem J. M., Brand L. Time-resolved fluorescence of proteins. Annu Rev Biochem. 1985;54:43–71. doi: 10.1146/annurev.bi.54.070185.000355. [DOI] [PubMed] [Google Scholar]
  3. Calhoun D. B., Vanderkooi J. M., Englander S. W. Penetration of small molecules into proteins studied by quenching of phosphorescence and fluorescence. Biochemistry. 1983 Mar 29;22(7):1533–1539. doi: 10.1021/bi00276a003. [DOI] [PubMed] [Google Scholar]
  4. Chang M. C., Cross A. J., Fleming G. R. Internal dynamics and overall motion of lysozyme studied by fluorescence depolarization of the eosin lysozyme complex. J Biomol Struct Dyn. 1983 Oct;1(1):299–318. doi: 10.1080/07391102.1983.10507441. [DOI] [PubMed] [Google Scholar]
  5. Cross A. J., Fleming G. R. Analysis of time-resolved fluorescence anisotropy decays. Biophys J. 1984 Jul;46(1):45–56. doi: 10.1016/S0006-3495(84)83997-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eftink M. R., Ghiron C. A. Dynamics of a protein matrix revealed by fluorescence quenching. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3290–3294. doi: 10.1073/pnas.72.9.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eftink M. R., Ghiron C. A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry. 1976 Feb 10;15(3):672–680. doi: 10.1021/bi00648a035. [DOI] [PubMed] [Google Scholar]
  8. Eftink M. Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins. Biophys J. 1983 Sep;43(3):323–334. doi: 10.1016/S0006-3495(83)84356-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grinvald A., Steinberg I. Z. The fluorescence decay of tryptophan residues in native and denatured proteins. Biochim Biophys Acta. 1976 Apr 14;427(2):663–678. doi: 10.1016/0005-2795(76)90210-5. [DOI] [PubMed] [Google Scholar]
  10. Heinemann U., Saenger W. Specific protein-nucleic acid recognition in ribonuclease T1-2'-guanylic acid complex: an X-ray study. Nature. 1982 Sep 2;299(5878):27–31. doi: 10.1038/299027a0. [DOI] [PubMed] [Google Scholar]
  11. Hershberger M. V., Maki A. H., Galley W. C. Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins. Biochemistry. 1980 May 13;19(10):2204–2209. doi: 10.1021/bi00551a032. [DOI] [PubMed] [Google Scholar]
  12. Inagaki F., Kawano Y., Shimada I., Takahashi K., Miyazawa T. Nuclear magnetic resonance study on the microenvironments of histidine residues of ribonuclease T1 and carboxymethylated ribonuclease T1. J Biochem. 1981 Apr;89(4):1185–1195. [PubMed] [Google Scholar]
  13. James D. R., Demmer D. R., Steer R. P., Verrall R. E. Fluorescence lifetime quenching and anisotropy studies of ribonuclease T1. Biochemistry. 1985 Sep 24;24(20):5517–5526. doi: 10.1021/bi00341a036. [DOI] [PubMed] [Google Scholar]
  14. Lakowicz J. R., Maliwal B. P., Cherek H., Balter A. Rotational freedom of tryptophan residues in proteins and peptides. Biochemistry. 1983 Apr 12;22(8):1741–1752. doi: 10.1021/bi00277a001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Osterman H. L., Walz F. G., Jr Subsite interactions and ribonuclease T1 catalysis: kinetic studies with APGpC and ApGpU. Biochemistry. 1979 May 15;18(10):1984–1988. doi: 10.1021/bi00577a021. [DOI] [PubMed] [Google Scholar]
  16. Pongs O. Influences of pH and substrate analogs on ribonuclease T1 fluorescence. Biochemistry. 1970 May 26;9(11):2316–2322. doi: 10.1021/bi00813a015. [DOI] [PubMed] [Google Scholar]
  17. Ross J. B., Schmidt C. J., Brand L. Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase. Biochemistry. 1981 Jul 21;20(15):4369–4377. doi: 10.1021/bi00518a021. [DOI] [PubMed] [Google Scholar]
  18. Sander C., Ts'o P. O. Circular dichroism studies on the conformation and interaction of T 1 ribonuclease. Biochemistry. 1971 May 25;10(11):1953–1966. doi: 10.1021/bi00787a001. [DOI] [PubMed] [Google Scholar]
  19. Sato S., Egami F. On the interaction of ribonuclease T-1 and guanosine 2'-phosphate and related compounds. Biochem Z. 1965 Aug 19;342(4):437–448. [PubMed] [Google Scholar]
  20. Sugio S., Oka K., Ohishi H., Tomita K., Saenger W. Three-dimensional structure of the ribonuclease T1 X 3'-guanylic acid complex at 2.6 A resolution. FEBS Lett. 1985 Apr 8;183(1):115–118. doi: 10.1016/0014-5793(85)80966-2. [DOI] [PubMed] [Google Scholar]
  21. TAKAHASHI K. The structure and function of ribonuclease T1. II. Further purification and amino acid composition of ribonuclease T1. J Biochem. 1962 Feb;51:95–108. doi: 10.1093/oxfordjournals.jbchem.a127515. [DOI] [PubMed] [Google Scholar]
  22. Takahashi K. A revision and confirmation of the amino acid sequence of ribonuclease T1. J Biochem. 1985 Sep;98(3):815–817. doi: 10.1093/oxfordjournals.jbchem.a135339. [DOI] [PubMed] [Google Scholar]
  23. Takahashi K. The amino acid sequence of ribonuclease T-1. J Biol Chem. 1965 Oct;240(10):4117–4119. [PubMed] [Google Scholar]
  24. Takahashi K. The structure and function of ribonuclease T1. 18. Gel filtration studies on the interaction of ribonuclease T1 with substrate analogs. J Biochem. 1972 Dec;72(6):1469–1481. doi: 10.1093/oxfordjournals.jbchem.a130039. [DOI] [PubMed] [Google Scholar]
  25. Turoverov K. K., Kuznetsova I. M., Zaitsev V. N. The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties. Biophys Chem. 1985 Nov;23(1-2):79–89. doi: 10.1016/0301-4622(85)80066-1. [DOI] [PubMed] [Google Scholar]
  26. Walz F. G., Jr, Hooverman L. L. Interaction of guanine ligands with ribonuclease T1. Biochemistry. 1973 Nov 20;12(24):4846–4851. doi: 10.1021/bi00748a006. [DOI] [PubMed] [Google Scholar]
  27. Walz F. G., Jr Studies on the nature of guanine nucleotide binding with ribonuclease T1. Biochemistry. 1977 Dec 13;16(25):5509–5515. doi: 10.1021/bi00644a018. [DOI] [PubMed] [Google Scholar]
  28. Walz F. G., Jr, Terenna B. Subsite interactions of ribonuclease T1: binding studies of dimeric substrate analogues. Biochemistry. 1976 Jun 29;15(13):2837–2842. doi: 10.1021/bi00658a021. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES