title: Picosecond fluorescence dynamics of tryptophan and 5-fluorotryptophan in monellin: slow water-protein relaxation unmasked.
abstract: Time dependent fluorescence Stokes (emission wavelength) shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluorescence probes, both the efficiency and the wavelength of Trp fluorescence in proteins are highly sensitive to microenvironment, and Stokes shifts can be dominated by the well-known heterogeneous nature of protein structure, leading to what we call pseudo-TDFSS: shifts that arise from differential decay rates of subpopulations. Here we emphasize a novel, general method that obviates pseudo-TDFSS by replacing Trp by 5-fluorotryptophan (5Ftrp), a fluorescent analogue with higher ionization potential and greatly suppressed electron-transfer quenching. 5FTrp slows and suppresses pseudo-TDFSS, thereby providing a clearer view of genuine relaxation caused by solvent and protein response. This procedure is applied to the sweet-tasting protein monellin which has uniquely been the subject of ultrafast studies in two different laboratories (Peon, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10964; Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214) that led to disparate interpretations of a 20 ps transient. They differed because of the pseudo-TDFSS present. The current study exploiting special properties of 5FTrp strongly supports the conclusion that both lifetime heterogeneity-based TDFSS and environment relaxation-based TDFSS are present in monellin and 5FTrp-monellin. The original experiments on monellin were most likely dominated by pseudo-TDFSS, whereas, in the present investigation of 5FTrp-monellin, the TDFSS is dominated by relaxation and any residual pseudo-TDFSS is overwhelmed and/or slowed to irrelevance.
authors.affiliation: †Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xu
authors.affiliation: †Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Binbin
authors.initials: B
authors.lastname: Chen
authors.forename: Patrik
authors.initials: P
authors.lastname: Callis
authors.affiliation: §Department of Chemistry, Saint Francis University, Loretto, Pennsylvania 15940, United States.
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Muiño
authors.affiliation: ∥Department of Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Henriëtte
authors.initials: H
authors.lastname: Rozeboom
authors.affiliation: ∥Department of Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
authors.affiliation: ⊥Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Toptygin
authors.affiliation: ⊥Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Brand
authors.affiliation: †Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Knutson
mesh_terms: D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014867:Water / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D014867:Water; C026927:5-fluorotryptophan; D014364:Tryptophan