Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1989 Jul;56(1):43–66. doi: 10.1016/S0006-3495(89)82651-7

Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence.

P H Axelsen 1, F G Prendergast 1
PMCID: PMC1280451  PMID: 2502198

Abstract

The interactions of tryptophan-59 (TRP-59) and its protein environment in ribonuclease-T1 (RNAse-T1) were examined in a 50-ps molecular dynamics simulation. The simulation used was previously shown to demonstrate a fluorescence anisotropy decay that closely agreed with the experimentally determined limiting anisotropy for RNAse-T1 (Axelsen, P. H., C. Haydock, and F. G. Prendergast. 1988. Biophys. J. 54:249-258). Further characterization of TRP-59 side chain dynamics and its protein environment has now been completed and correlated to other photophysical properties of this protein. Angular fluctuations of the side chain occur at rates of 1-10 cycles/ps and are limited to +/- 0.3 radians in all directions. Side chain motions are primarily limited by nonpolar collisions, although most side chain atoms have some collisional contact with polar atoms in the adjacent protein matrix or water. The steric relationship between PRO-39 and TRP-59 changes abruptly at 16 ps into the simulation. Two types of interaction with water are observed. First, a structural water appears to H-bond with the greater than N-H group of TRP-59. Second, water frequently contacts the six-atom ring. The electrostatic field experienced by the TRP-59 rings appears to be relatively constant and featureless regardless of ring orientation. We make the following interferences from our data: The fluorescent emission of TRP-59 may be red-shifted relative to TRP in nonpolar solvents either as a result of specific interactions with the structural water or relaxations of proximal bulk water and polar protein moieties. The quenching efficiency of polar interactions with TRP-59 must be extremely low given their frequency and the high quantum yield of RNAse-T1. This low efficiency may be due to restricted and unfavorable interaction geometries. PRO-39 is located near two titratable HIS residues in RNAse-T1 and may be involved in pH-dependent fluorescence phenomena by virtue of a metastable interaction with TRP-59. The interaction of bulk water with TRP-59 illustrates features of the gated transition state model for transient exposure to exogenously added collisional quenching agents. The restrictive environment of TRP-59 suggests that extrinsic quenching can only occur via interactions with the edge of the indole six-atom ring and that the efficiency of a quencher in a protein environment is likely to be a function of molecular symmetry.

Full text

PDF
43

Images in this article

50FIGURE 4
on p.50
51FIGURE 5
on p.51
54FIGURE 8
on p.54

Selected References

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

  1. Alcala J. R., Gratton E., Prendergast F. G. Fluorescence lifetime distributions in proteins. Biophys J. 1987 Apr;51(4):597–604. doi: 10.1016/S0006-3495(87)83384-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alcala J. R., Gratton E., Prendergast F. G. Interpretation of fluorescence decays in proteins using continuous lifetime distributions. Biophys J. 1987 Jun;51(6):925–936. doi: 10.1016/S0006-3495(87)83420-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Axelsen P. H., Haydock C., Prendergast F. G. Molecular dynamics of tryptophan in ribonuclease-T1. I. Simulation strategies and fluorescence anisotropy decay. Biophys J. 1988 Aug;54(2):249–258. doi: 10.1016/S0006-3495(88)82954-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bash P. A., Singh U. C., Brown F. K., Langridge R., Kollman P. A. Calculation of the relative change in binding free energy of a protein-inhibitor complex. Science. 1987 Jan 30;235(4788):574–576. doi: 10.1126/science.3810157. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  8. Burstein E. A., Permyakov E. A., Yashin V. A., Burkhanov S. A., Finazzi Agro A. The fine structure of luminescence spectra of azurin. Biochim Biophys Acta. 1977 Mar 28;491(1):155–159. doi: 10.1016/0005-2795(77)90051-4. [DOI] [PubMed] [Google Scholar]
  9. Burstein E. A., Vedenkina N. S., Ivkova M. N. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973 Oct;18(4):263–279. doi: 10.1111/j.1751-1097.1973.tb06422.x. [DOI] [PubMed] [Google Scholar]
  10. Chen L. X., Longworth J. W., Fleming G. R. Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study. Biophys J. 1987 Jun;51(6):865–873. doi: 10.1016/S0006-3495(87)83414-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Connolly M. L. Solvent-accessible surfaces of proteins and nucleic acids. Science. 1983 Aug 19;221(4612):709–713. doi: 10.1126/science.6879170. [DOI] [PubMed] [Google Scholar]
  12. Cotton F. A., Hazen E. E., Jr, Legg M. J. Staphylococcal nuclease: proposed mechanism of action based on structure of enzyme-thymidine 3',5'-bisphosphate-calcium ion complex at 1.5-A resolution. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2551–2555. doi: 10.1073/pnas.76.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Eftink M. R., Ghiron C. A. Frequency domain measurements of the fluorescence lifetime of ribonuclease T1. Biophys J. 1987 Sep;52(3):467–473. doi: 10.1016/S0006-3495(87)83235-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Ghiron C. A., Longworth J. W. Transfer of singlet energy within trypsin. Biochemistry. 1979 Aug 21;18(17):3828–3832. doi: 10.1021/bi00584a029. [DOI] [PubMed] [Google Scholar]
  18. Ghosh I., McCammon J. A. Sidechain rotational isomerization in proteins. Dynamic simulation with solvent surroundings. Biophys J. 1987 Apr;51(4):637–641. doi: 10.1016/S0006-3495(87)83388-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gilson M. K., Honig B. H. Calculation of electrostatic potentials in an enzyme active site. Nature. 1987 Nov 5;330(6143):84–86. doi: 10.1038/330084a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gryczynski I., Eftink M., Lakowicz J. R. Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1. Biochim Biophys Acta. 1988 Jun 13;954(3):244–252. doi: 10.1016/0167-4838(88)90079-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Henry E. R., Hochstrasser R. M. Molecular dynamics simulations of fluorescence polarization of tryptophans in myoglobin. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6142–6146. doi: 10.1073/pnas.84.17.6142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Ilich P., Axelsen P. H., Prendergast F. G. Electronic transitions in molecules in static external fields. I. Indole and Trp-59 in ribonuclease T1. Biophys Chem. 1988 Apr;29(3):341–349. doi: 10.1016/0301-4622(88)85056-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lakowicz J. R., Laczko G., Gryczynski I., Cherek H. Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry. J Biol Chem. 1986 Feb 15;261(5):2240–2245. [PubMed] [Google Scholar]
  28. Lakowicz J. R., Laczko G., Gryczynski I. Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry. Biochemistry. 1987 Jan 13;26(1):82–90. doi: 10.1021/bi00375a012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  31. Leszczynski J. F., Rose G. D. Loops in globular proteins: a novel category of secondary structure. Science. 1986 Nov 14;234(4778):849–855. doi: 10.1126/science.3775366. [DOI] [PubMed] [Google Scholar]
  32. MacKerell A. D., Jr, Nilsson L., Rigler R., Saenger W. Molecular dynamics simulations of ribonuclease T1: analysis of the effect of solvent on the structure, fluctuations, and active site of the free enzyme. Biochemistry. 1988 Jun 14;27(12):4547–4556. doi: 10.1021/bi00412a049. [DOI] [PubMed] [Google Scholar]
  33. MacKerell A. D., Jr, Rigler R., Nilsson L., Hahn U., Saenger W. Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1. Biophys Chem. 1987 May 9;26(2-3):247–261. doi: 10.1016/0301-4622(87)80027-3. [DOI] [PubMed] [Google Scholar]
  34. Petrich J. W., Longworth J. W., Fleming G. R. Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins. Biochemistry. 1987 May 19;26(10):2711–2722. doi: 10.1021/bi00384a010. [DOI] [PubMed] [Google Scholar]
  35. Ringe D., Petsko G. A. Mapping protein dynamics by X-ray diffraction. Prog Biophys Mol Biol. 1985;45(3):197–235. doi: 10.1016/0079-6107(85)90002-1. [DOI] [PubMed] [Google Scholar]
  36. Russell S. T., Warshel A. Calculations of electrostatic energies in proteins. The energetics of ionized groups in bovine pancreatic trypsin inhibitor. J Mol Biol. 1985 Sep 20;185(2):389–404. doi: 10.1016/0022-2836(85)90411-5. [DOI] [PubMed] [Google Scholar]
  37. Somogyi B., Norman J. A., Rosenberg A. Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins. An extended model. Biophys J. 1986 Jul;50(1):55–61. doi: 10.1016/S0006-3495(86)83438-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Van Belle D., Couplet I., Prevost M., Wodak S. J. Calculations of electrostatic properties in proteins. Analysis of contributions from induced protein dipoles. J Mol Biol. 1987 Dec 20;198(4):721–735. doi: 10.1016/0022-2836(87)90213-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES