We report measurements of fluorescence quantum yields of tryptophan, tryptophanylaspartate and tryptophanylarginine in several solvents as well as in aqueous solutions over a wide range of pH. We aim to test a computational model developed by Callis and coworkers of fluorescence quantum yield, which postulates that quenching in tryptophan arises from energy loss due to an electron transfer from the aromatic system of tryptophan to one of the amides in the protein backbone. Since the electron transfer state is expected to be high in energy, normally this would not be a possible outcome, but because of its large dipole, such a state should be more accessible in polar solvents. In addition, conditions of low (high) pH, which result in a net positive (negative) charge for the terminal amine (carboxyl) should result in an increase (decrease) of electron transfer rates and low (high) quantum yields. The observed results confirm the predictions of the model.
KeywordsTryptophanFluorescenceQuenchingElectron Transfer
Demchenko, A.P. (1986) Ultrasiolet spectroscopy of proteins. Springer-Verlag, New York.
Eftink, M.R. (1991) Fluorescence techniques for studying protein structure. Methods of Biochemical Analysis, 35, 127-205. doi:10.1002/9780470110560.ch3
Weber, G. (1960) Fluorescence-polarization spectrum and electronic energy transfer in tyrosine, tryptophan and related compounds. Biochemical Journal, 75, 335-345.
Konev, S.V. (1967) Fluorescence and phosphorescence of proteins and nucleic acids. Plenum, New York.
Beechem, J.M. and Brand, L. (1985) Time-resolved fluorescence of proteins. Annual Review of Biochemistry, 54, 43-71. doi:10.1146/annurev.bi.54.070185.000355
Muí?o, P.L. and Callis, P.R. (1994) Hybrid simulations of salvation effects on electronic spectra: Indoles in water. Journal of Chemical Physics, 100, 4093-4109.
Callis, P.R. (1997) 1La and 1Lb transitions of tryptophan: Applications of theory and experimental observations to fluorescence of proteins. Methods in Enzymology, 278, 113-150. doi:10.1016/S0076-6879(97)78009-1
Chen Y. and Barkley, M.D. (1998) Toward understanding tryptophan fluorescence in proteins. Biochemistry, 37, 9976-9982. doi:10.1021/bi980274n
Vivian, J.T. and Callis, P.R. (2001) Mechanisms of tryptophan fluorescence shifts in proteins. Biophysical Journal, 80, 2093-2109. doi:10.1016/S0006-3495(01)76183-8
Toptygin, D., Savtchenko, R.S., Meadow, N.D. and Brand, L. (2001) Homogeneous spectrally- and time-resolved fluorescence emission from single-tryptophan mutants of IIAGlc. Journal of Physical Chemistry B, 105, 2043-2055. doi:10.1021/jp003405e
Xu, J., Chen, J., Toptygin, D., Tcherkasskaya, O., Callis, P.R., King, J., Brand, L. and Knutson, J.R. (2009) Femtosecond fluorescence spectra of tryptophan in human γ-crystallin mutants: Site-dependent ultrafast quenching. Journal of the American Chemical Society, 131, 16751- 16757. doi:10.1021/ja904857t
Cowgill, R.W. (1970) Fluorescence and protein structure. XVII. On the mechanism of peptide quenching. Biochimica et Biophysica Acta, 200, 18-25.
Feitelson, J. (1970) Environmental effects on the fluorescence of tryptophan and other indole derivatives. Israel Journal of Chemistry, 8, 241-252.
Petrich, J.W., Chang, M.C., McDonald, D.D. and Fleming, G.R. (1983) On the origin of nonexponential fluorescence decay in tryptophan and its derivatives. Journal of the American Chemical Society, 105, 3824-3832. doi:10.1021/ja00350a014
Chen, Y., Liu, B., Yu, H.T., Barkley, M.D. (1996) The peptide bond quenches indole fluorescence. Journal of the American Chemical Society, 118, 9271-9278. doi:10.1021/ja961307u
Sillen, A., Hennecke, J., Roethlisberger, D., Glockshuber, R. and Engelborghs, Y. (1999) Fluorescence quenching in the DsbA protein from E. coli: Complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan. Proteins: Structure, Function, and Genetics. 37, 253-263. doi:10.1002/(SICI)1097-0134(19991101)37:2 3.0.CO;2-J
Vivian, J.T. and Callis, P.R. (2002) Understanding the variable fluorescence quantum yield of tryptophan in proteins using QM-MM simulations. Quenching by charge transfer to the peptide backbone. Chemical Physics Letters, 369, 409-414.
Callis, P.R., Liu, T. (2004) Quantitative prediction of fluorescence quantum yields for tryptophan in proteins. Journal of Physical Chemistry B, 108, 4248-4259. doi:10.1021/jp0310551
Callis, P.R., Petrenko, A., Muí?o, P.L., Tusell, J.R. (2007) Ab Initio prediction of tryptophan fluorescence quenching by protein electric field-enabled electron transfer. Journal of Physical Chemistry B, 111, 10335-10339. doi:10.1021/jp0744883
Muí?o, P.L. and Callis, P.R. (2009) Solvent effects on the fluorescence quenching of tryptophan by amides via electron transfer. Experimental and computational studies. Journal of Physical Chemistry B, 113, 2572-2577. doi:10.1021/jp711513b
Pan, C.P., Muí?o, P.L., Barkley, M.D. and Callis, P.R. (2011) Correlation of tryptophan fluorescence spectral shifts and lifetimes arising directly from heterogeneous environment. Journal of Physical Chemistry B, 115, 3245-3253. doi:10.1021/jp111925w
Chen, R., Knutson, J.R., Ziffer, H. and Porter, D. (1991) Fluorescence of tryptophan dipeptides: Correlations with the rotamer model. Biochemistry, 30, 5184-5195. doi:10.1021/bi00235a011
Xu, J. and Knutson, J.R. (2009) Quasi-static self- quenching of Trp-X and X-Trp dipeptides in water: Ultrafast fluorescence decay. Journal of Physical Chemistry B, 113, 12084-12089. doi:10.1021/jp903078x
Yu, H.T., Colucci, W.J., McLaughlin, M.L. and Barkley, M.D. (1992) Fluorescence quenching of indoles by excited state proton transfer. Journal of the American Chemical Society, 114, 8449-8454. doi:10.1021/ja00048a015
Eftink, M.R., Jia, Y., Hu, D. and Ghiron, J.A. (1995) Fluorescence studies with tryptophan analogs: Excited state interactions involving the side chain amino group. Journal of Physical Chemistry, 99, 5713-5723. doi:10.1021/j100015a064
Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K. M. (1986) Data for biochemical research. Clarendon Press, Oxford.
Blancafort, L., González, D., Olivucci, M., Robb, M.A. (2002) Quenching of tryptophan 1(π,π*) fluorescence induced by intramolecular hydrogen abstraction via an aborted decarboxylation mechanism. Journal of the American Chemical Society, 124, 6398-6406. doi:10.1021/ja016915a
Sobolewski, A.L. and Domcke, W. (1999) Ab initio investigations on the photophysics of indole. Chemical Physics Letters. 315, 293-298. doi:10.1016/S0009-2614(99)01249-X