(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

I.H.M. van Stokkum, B. Gobets, T. Gensch, F. van Mourik, K.J. Hellingwerf, R. van Grondelle, J.T.M. Kennis

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3-carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes. © 2006 American Society for Photobiology.
Original languageEnglish
Pages (from-to)380-388
JournalPhotochemistry and Photobiology
Volume82
Issue number2
DOIs
Publication statusPublished - 2006

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Streak cameras
streak cameras
Chromophores
Fluorescence
proteins
life (durability)
fluorescence
Proteins
decay
Photobiology
chromophores
Bacteriorhodopsins
Photochemistry
Coumaric Acids
Photochemical reactions
Signal-To-Noise Ratio
Isomerization
Green Fluorescent Proteins
time constant
Quenching

Bibliographical note

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

Cite this

@article{632b28e5afa14e7d9226bd653fc6a745,
title = "(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system",
abstract = "The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3-carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes. {\circledC} 2006 American Society for Photobiology.",
author = "{van Stokkum}, I.H.M. and B. Gobets and T. Gensch and {van Mourik}, F. and K.J. Hellingwerf and {van Grondelle}, R. and J.T.M. Kennis",
note = "(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system",
year = "2006",
doi = "10.1562/2005-06-15-RA-572",
language = "English",
volume = "82",
pages = "380--388",
journal = "Photochemistry and Photobiology",
issn = "0031-8655",
publisher = "Wiley-Blackwell",
number = "2",

}

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system. / van Stokkum, I.H.M.; Gobets, B.; Gensch, T.; van Mourik, F.; Hellingwerf, K.J.; van Grondelle, R.; Kennis, J.T.M.

In: Photochemistry and Photobiology, Vol. 82, No. 2, 2006, p. 380-388.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - (Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

AU - van Stokkum, I.H.M.

AU - Gobets, B.

AU - Gensch, T.

AU - van Mourik, F.

AU - Hellingwerf, K.J.

AU - van Grondelle, R.

AU - Kennis, J.T.M.

N1 - (Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

PY - 2006

Y1 - 2006

N2 - The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3-carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes. © 2006 American Society for Photobiology.

AB - The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3-carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes. © 2006 American Society for Photobiology.

U2 - 10.1562/2005-06-15-RA-572

DO - 10.1562/2005-06-15-RA-572

M3 - Article

VL - 82

SP - 380

EP - 388

JO - Photochemistry and Photobiology

JF - Photochemistry and Photobiology

SN - 0031-8655

IS - 2

ER -