The structural basis for spectral variations in green fluorescent protein

Palm, Gottfried J.; Zdanov, Alexander; Gaitanaris, George A.; Stauber, Roland H.; Pavlakis, George N.; Wlodawer, Alexander

doi:10.1038/nsb0597-361

Cited by 249 publications

(304 citation statements)

References 20 publications

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“…The bulge is centered on His 148 , the side chain of which partially fills the bulge and interacts with the chromophore phenol(ate) oxygen (34,51,56) (see Fig. 2A).…”

Section: Discussionmentioning

confidence: 99%

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

Hanson

Aggeler

Oglesbee

et al. 2004

Journal of Biological Chemistry

901

957

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Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0-and 1.9-Å resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near ؊360 mV (assuming mitochondrial pH ϳ8.0 at 37°C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

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“…The bulge is centered on His 148 , the side chain of which partially fills the bulge and interacts with the chromophore phenol(ate) oxygen (34,51,56) (see Fig. 2A).…”

Section: Discussionmentioning

confidence: 99%

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

Hanson

Aggeler

Oglesbee

et al. 2004

Journal of Biological Chemistry

901

957

View full text Add to dashboard Cite

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“…It was postulated that the molecular ground state of the chromophore in this anionic form acts as an intermediate in the GFP photocycle (2,(5)(6)(7)(8). However, with time-resolved spectroscopy, only the excited states A* and I* of GFP could be detected (9), and the molecular nature and dynamics of any ground-state intermediate remained elusive.…”

mentioning

confidence: 99%

Uncovering the hidden ground state of green fluorescent protein

Kennis

Larsen²,

Stokkum³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

141

232

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The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I 1 and I2, has been unveiled. I1 and I2 absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I 2 intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen͞deuterium exchange of the protein leads to a significant increase of the I1 and I2 lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.hidden reaction intermediates ͉ multipulse control spectroscopy ͉ proton transfer ͉ ultrafast spectroscopy

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“…It is currently unclear whether this is the same I form observed in the time-resolved fluorescence experiments. Although the I form has so far not been described in atomic detail, a structural model of I was suggested on the basis of comparisons between the crystal structures of wild-type (A form) and mutant GFP proteins containing the B form (7,8); see Fig. 2.…”

mentioning

confidence: 99%

Proton shuttle in green fluorescent protein studied by dynamic simulations

Lill

Helms

2002

Proc. Natl. Acad. Sci. U.S.A.

138

136

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As a direct simulation of a multistep proton transfer reaction involving protein residues, the proton relay shuttle between A and I forms of green fluorescent protein (GFP) is simulated in atomic detail by using a special molecular dynamics simulation technique. Electronic excitation of neutral chromophore in wild-type GFP is generally followed by excited-state proton transfer to a nearby glutamic acid residue via a water molecule and a serine residue. Here we show that the second and third transfer steps occur ultrafast on time scales of several tens of femtoseconds. Proton back-shuttle in the ground state is slower and occurs in a different sequence of events. The simulations provide atomic models of various intermediates and yield realistic rate constants for proton transfer events. In particular, we argue that the I form observed spectroscopically under equilibrium conditions may differ from the I form observed as a fast intermediate by an anti to syn rotation of the carboxyl proton of neutral Glu-222.B ecause of its unique photophysical properties-strong green fluorescence without need for an additional cofactorgreen fluorescent protein (GFP) has become a very powerful marker for gene expression, cellular localization, and dynamic intracellular events over the past 5 years (1). Its rich photophysical behavior was characterized quite well by a great variety of spectroscopic techniques (1-4), and its three-dimensional structure was determined by x-ray crystallography (5, 6). Wild-type GFP exhibits two absorption maxima ''A'' and ''B'' at 395 nanometers (nm) and at 475 nm (1) that are present roughly in a 6:1 ratio (1), and that correspond to forms of the protein with either neutral (A) or anionic (B) chromophores. Time-resolved fluorescence spectroscopy identified an additional form ''I*'' that is populated as a fast intermediate after excitation of the A form, A* 3 I* (2, 3). The mechanism of interconversion is likely caused by excited-state proton transfer from the chromophore to a nearby protein residue (2, 3) because the new form emits at a similar wave length as B* and because the transfer rate slows down upon deuteration (2); see Fig. 1 for an overview.Although ''I form'' originally denoted only this fast intermediate, it has become common to use ''I form'' as a term for GFP species that absorb at wavelengths red-shifted with respect to the B form. Surprisingly, recent hole-burning experiments demonstrated that an I form is already present in wild-type GFP samples at room temperature under equilibrium conditions (4). It is currently unclear whether this is the same I form observed in the time-resolved fluorescence experiments. Although the I form has so far not been described in atomic detail, a structural model of I was suggested on the basis of comparisons between the crystal structures of wild-type (A form) and mutant GFP proteins containing the B form (7, 8); see Fig. 2. In this model, Glu-222 is assumed to be protonated in the I form (7), thus requiring a rise of the pK a of Glu-222 by more than 4 ...

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The structural basis for spectral variations in green fluorescent protein

Cited by 249 publications

References 20 publications

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators

Uncovering the hidden ground state of green fluorescent protein

Proton shuttle in green fluorescent protein studied by dynamic simulations

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