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. 1989 Mar;55(3):575–579. doi: 10.1016/S0006-3495(89)82851-6

Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

M R Eftink 1, C A Ghiron 1, R A Kautz 1, R O Fox 1
PMCID: PMC1330511  PMID: 2649165

Abstract

Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures.

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Selected References

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  1. Alcala J. R., Gratton E., Prendergast F. G. Resolvability of fluorescence lifetime distributions using phase fluorometry. Biophys J. 1987 Apr;51(4):587–596. doi: 10.1016/S0006-3495(87)83383-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnone A., Bier C. J., Cotton F. A., Day V. W., Hazen E. E., Jr, Richardson D. C., Yonath A., Richardson J. S. A high resolution structure of an inhibitor complex of the extracellular nuclease of Staphylococcus aureus. I. Experimental procedures and chain tracing. J Biol Chem. 1971 Apr 10;246(7):2302–2316. [PubMed] [Google Scholar]
  3. 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]
  4. Calderon R. O., Stolowich N. J., Gerlt J. A., Sturtevant J. M. Thermal denaturation of staphylococcal nuclease. Biochemistry. 1985 Oct 22;24(22):6044–6049. doi: 10.1021/bi00343a004. [DOI] [PubMed] [Google Scholar]
  5. Castelli F., White H. D., Forster L. S. Lifetime and quenching of tryptophan fluorescence in whiting parvalbumin. Biochemistry. 1988 May 3;27(9):3366–3372. doi: 10.1021/bi00409a037. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Evans P. A., Dobson C. M., Kautz R. A., Hatfull G., Fox R. O. Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis. Nature. 1987 Sep 17;329(6136):266–268. doi: 10.1038/329266a0. [DOI] [PubMed] [Google Scholar]
  9. Fox R. O., Evans P. A., Dobson C. M. Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy. Nature. 1986 Mar 13;320(6058):192–194. doi: 10.1038/320192a0. [DOI] [PubMed] [Google Scholar]
  10. Gratton E., Limkeman M. A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys J. 1983 Dec;44(3):315–324. doi: 10.1016/S0006-3495(83)84305-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Lakowicz J. R., Cherek H., Gryczynski I., Joshi N., Johnson M. L. Analysis of fluorescence decay kinetics measured in the frequency domain using distributions of decay times. Biophys Chem. 1987 Oct;28(1):35–50. doi: 10.1016/0301-4622(87)80073-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lakowicz J. R., Joshi N. B., Johnson M. L., Szmacinski H., Gryczynski I. Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays. J Biol Chem. 1987 Aug 15;262(23):10907–10910. [PubMed] [Google Scholar]
  15. 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]
  16. Laws W. R., Ross J. B., Wyssbrod H. R., Beechem J. M., Brand L., Sutherland J. C. Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics. Biochemistry. 1986 Feb 11;25(3):599–607. doi: 10.1021/bi00351a013. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Szabo A. G., Stepanik T. M., Wayner D. M., Young N. M. Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study. Biophys J. 1983 Mar;41(3):233–244. doi: 10.1016/S0006-3495(83)84433-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

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