Ultrafast Dielectric Response of Proteins from Dynamics Stokes Shifting of Coumarin in Calmodulin

Changenet‐Barret, Pascale; Choma, Christin T.; Gooding, E.; DeGrado, William F.; Hochstrasser, Robin M.

doi:10.1021/jp001634v

Cited by 118 publications

(141 citation statements)

References 57 publications

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“…The simulation of a single Trp in proteins under the partial exposure to water gave a time scale of several picoseconds for the decay of the shifted spectral components because of the large-amplitude motions of the protein backbone and side chains and͞or wholesale rearrangement of nearby hydrogen-bonded water clusters, consistent with our observation of solvation occurring up to 10 ps or longer. By placing an extrinsic dye probe in a protein pocket, solvation dynamics of polar amino acid residues or rigid water molecules on slower time scales also have been recently reported (25,26). Ultrafast solvation dynamics in protein by using the intrinsic probe, Trp, were not reported before.…”

Section: Resultsmentioning

confidence: 98%

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Zhong

Pal

Zhang

et al. 2001

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

135

134

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We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution (Ϸ160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15-20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R 0) was determined, by using the known x-ray data, to be 19.5 Å for Trp-36 and 25.2 Å for Trp-3, respectively. The orientation factor ( 2 ) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with 2 value of 2͞3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.T ryptophan (Trp) is the most important fluorophore among amino acid residues for optical probing of proteins. However, Trp fluorescence is complex because of different rotamers in the ground state and the two nearly degenerate electronic states ( 1 L a , 1 L b ) with perpendicular transition moments. Accordingly, numerous studies (1-10) have focused on the lifetime, quantum yield, Stokes shift, and fluorescence anisotropy. Most of these studies were made with picosecond or nanosecond time resolution (3, 6-10). To probe the local protein dynamics, Trp solvation by neighboring soft͞rigid water molecules, or by other polar amino acid residues, must be resolved on the femtosecond time scale. Moreover, such studies are important for examining the nature of resonance energy transfer (RET) that is used for deducing distances and orientations between the Trp and quenchers in the protein (e.g., see refs. 3 and 10, and references therein).We choose the hyperthermophilic iron-sulfur protein, Pyrococcus furiosus rubredoxin (PfRd), as a prototype system (Fig. 1). The high-resolution x-ray crystallographic structures of PfRd at 0.95 Å and its formylmethionine variant (fmetPfRd) at 1.2 Å, have been recently reported (11). PfRd is a small protein of 53 amino acid residues with an iron a...

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Section: Resultsmentioning

confidence: 98%

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Zhong

Pal

Zhang

et al. 2001

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

135

134

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“…Application of Multipulse Control Spectroscopy to GFP. The stimulated emission process can be used to transfer population on a femtosecond timescale from the excited state to the ground state: with an intense pulse resonant with the stimulated emission, a portion of the excited-state population can be deexcited or ''dumped'' to the ground state (11,12,22). In the case of GFP, this is achieved by applying an additional green pulse in the time-resolved experiment.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, it shows a diminished B band, allowing for a higher selectivity of A band excitation with near-UV light. Multipulse spectroscopy can be used to control and influence the course of reactions as they evolve, and its power lies in the ability to selectively remove or transfer the population of transient species with carefully timed laser pulses (11)(12)(13). We have recently expanded this technique by combining it with broad-band spectral probing and elaborate global analysis techniques, enabling us to disentangle complex branched reaction schemes (14)(15)(16)(17)(18).…”

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.

140

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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“…While DANCA and ANSDMA are both capable of charge transfer in the excited state, coumarin 153 is exquisitely inert, which is one of the reasons it has been so extensively employed as a probe of solvation. 57,[62][63][64][65][66][67][68][69][70][71][72][73] (The DANCA experiments used an excitation wavelength of 345 nm. Novaira et al have observed that both LE and CT emission can be observed in reverse micelles at 330 nm.…”

Section: L(t) )mentioning

confidence: 99%

Solvation Dynamics of the Fluorescent Probe PRODAN in Heterogeneous Environments: Contributions from the Locally Excited and Charge-Transferred States

2009

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The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,Ndimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/ water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and ∼0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states. ReceiVed: June 1, 2009; ReVised Manuscript ReceiVed: July 20, 2009 The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and ∼0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states.

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Ultrafast Dielectric Response of Proteins from Dynamics Stokes Shifting of Coumarin in Calmodulin

Cited by 118 publications

References 57 publications

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Uncovering the hidden ground state of green fluorescent protein

Solvation Dynamics of the Fluorescent Probe PRODAN in Heterogeneous Environments: Contributions from the Locally Excited and Charge-Transferred States

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