Subpicosecond fluorescence anisotropy studies of tryptophan in water

Ruggiero, Anthony J.; Todd, D.; Fleming, Graham R.

doi:10.1021/ja00159a017

Cited by 119 publications

(108 citation statements)

References 3 publications

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“…Furthermore, the estimated initial anisotropy is far below the theoretical limit of 0.4, 28 which is the expected value before electronic equilibration and solvation take place. The equilibration between 1 L a and 1 L b is known to result in a nonrotational contribution to the anisotropy decay of 2 ps, 6 leading to strong depolarization that cannot be resolved in this experiment. Spectral relaxation is ruled out because this process should result in a red shift of the emission, 11 whereas in this case the opposite is observed.…”

Section: Discussionmentioning

confidence: 87%

Ultrafast Polarized Fluorescence Measurements on Tryptophan and a Tryptophan-Containing Peptide

et al. 2003

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In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan and tryptophan in a small 22-mer peptide using a streak camera coupled to a spectrograph as a detection system. In both cases the fluorescence decay was multiexponential with decay times of ∼500 ps and ∼4 ns. Surprisingly, also a short-lived (∼16 ps) isotropic fluorescence component was found for both tryptophan and the peptide which, to our knowledge, has never been observed before. Fluorescence anisotropy data showed rotational correlation times of 155 ps and 1.5 ns for the peptide, reflecting local and overall peptide dynamics, respectively. Recently it was argued [Lakowicz, J. R. Photochem. Photobiol. 2000, 72, 421] that the nonexponential fluorescence decay of tryptophan in proteins is mainly due to spectral relaxation, whereas for isolated tryptophan it is due to different rotamers. Our results do not support this view. In contrast we conclude in both cases that the different fluorescence decay times should be ascribed to different rotameric states.

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Section: Discussionmentioning

confidence: 87%

Ultrafast Polarized Fluorescence Measurements on Tryptophan and a Tryptophan-Containing Peptide

et al. 2003

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“…Anisotropy and Internal Conversion. The initial anisotropy of Trp, ''r 0 '', has been extensively studied and discussed (3,4,8,39,40). In brief, it shows strong dependence on excitation wavelength.…”

Section: Resultsmentioning

confidence: 99%

“…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)(2)(3)(4)(5)(6)(7)(8)(9)(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)(7)(8)(9)(10).…”

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confidence: 99%

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.

138

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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“…1 L b is the lowest S 1 state, equilibration between these two states is believed to be very fast (of the order of 10 -12 sec), so that emission only from the lower S 1 state is observed. 56 It is generally believed that 1 L a is the fluorescing state in all proteins with the possible exception of Trp-48 of azurin. 55 In molecular terms, tryptophan is a unique amino acid since it is capable of both hydrophobic and polar interactions.…”

Section: Intrinsic Fluorescence Of Proteins and Peptides: Tryptophan mentioning

confidence: 99%

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

Raghuraman

Kelkar

Chattopadhyay

Reviews in Fluorescence 2005

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A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a corresponding shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which depends on the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES extremely useful since hydration plays a crucial modulatory role in the formation and maintenance of organized molecular assemblies such as folded proteins in aqueous solutions and biological membranes. The application of REES as a powerful tool to monitor the organization and dynamics of a variety of soluble, cytoskeletal, and membrane-bound proteins is discussed.

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Subpicosecond fluorescence anisotropy studies of tryptophan in water

Cited by 119 publications

References 3 publications

Ultrafast Polarized Fluorescence Measurements on Tryptophan and a Tryptophan-Containing Peptide

Ultrafast Polarized Fluorescence Measurements on Tryptophan and a Tryptophan-Containing Peptide

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

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

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