Fluorescence decay of tryptophan conformers in aqueous solution

Szabo, Arthur G.; Rayner, D. M.

doi:10.1021/ja00522a020

Cited by 573 publications

(455 citation statements)

References 12 publications

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“…We have used the spectrum at 20 ps to define the max (ϱ) value, because the spectral evolution related to solvation is completed by this time (16,17), whereas the nanosecond spectral change occurs because of the different fluorescence lifetimes of the two rotamers of Trp (26). In this and the rest of our measurements, we did not include wavelengths where a significant contribution from Raman scattering by water was evident (about Ϯ 5 nm, Ϸ3,400 cm Ϫ1 OH band from the respective excitation wavelength).…”

Section: Resultsmentioning

confidence: 99%

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Pal

Peón

Zewail

2002

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

528

578

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Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein-protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin Carlsberg, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of Ϸ7 Å and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (Ϸ1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of Ϸ7 Å from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.W ater is essential for the stability and function of biological macromolecules, proteins and DNA. Hydration plays a major role in the assembly of a protein's structure and dynamics. For example, water molecules around hydrophobic and hydrophilic sites are important to the understanding of the activity of enzyme proteins (see, e.g., refs. 1-3) and are part of the recognition process by other molecules or proteins. The water molecules that make up the hydration shell in the immediate vicinity of the surface are particularly relevant to the function and, in that sense, are termed biological water; this distinction has been discussed clearly by Nandi and Bagchi (4) in relation to dielectric relaxations. The nature of this shell ''layer'' has been the focus of numerous studies both theoretically and experimentally (see refs. 5-12), yet there is no generalized picture of the dynamics at the local molecular level.X-ray crystallography, neutron diffraction, and molecular dynamics studies have shown (5-10) that at protein surfaces, water molecules are site-selective and can be restricted in their motion, even existing in the form of clusters in some cases. For example, neutron diffraction experiments (9) followed by molecular dynamics simulations on carboxymyoglobin (10) revealed that among the 89 water molecules associated with the protein, 4 remain bound during the entire length of the molecular dynamics simulation (50 ps), whereas ...

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Section: Resultsmentioning

confidence: 99%

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Pal

Peón

Zewail

2002

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

528

578

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“…27,28 Several mechanisms were proposed to explain the observed heterogeneity of the Trp lifetime in single Trp proteins including the presence of Trp side chain rotamers, quenching by water molecules, electron transfer to the peptide carbonyl group, excited state electron or proton transfer and intersystem crossing. [29][30][31] Interestingly, an overlay of the 15 lowest energy structures of Ca 21 bound DREAM determined by NMR spectroscopy 22 and the structure of the DREAM C-terminal domain (residues 161-256) 23 displays the presence of a single Trp rotamer (t rotamer) in the DREAM structure. However, as shown in Figure 7, a transition from the t to g1 rotamer of Trp169 sidechain is observed during molecular dynamic simulations, which support the idea that the bimodal distribution could arise from two rotameric orientations of Trp169 with the side chain of the g1 rotamer being more solvent exposed (see below).…”

Section: Discussionmentioning

confidence: 99%

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

et al. 2015

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Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca 21 concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca 21 /Mg 21 association to DREAM. Our data indicate a minor impact of Ca 21 association on the overall structure of the Nand C-terminal domains, although Ca 21 binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca 21 bound forms of the protein. Time-resolved fluorescence data indicate that Ca 21 binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca 21 association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca 21 and Mg 21 suggesting that, under physiological conditions, Ca 21 free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

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“…The two slower isotropic times are similar to the 0.51 ns and 3.1 ns obtained by Szabo and Reyner. 22 These two lifetimes were attributed to different rotameric states of Trp. Estimates of the spectra for these different conformers were obtained earlier, measuring the fluorescence decay of Trp at different wavelengths (e.g., ref 22).…”

Section: Discussionmentioning

confidence: 99%

“…22 These two lifetimes were attributed to different rotameric states of Trp. Estimates of the spectra for these different conformers were obtained earlier, measuring the fluorescence decay of Trp at different wavelengths (e.g., ref 22). However, with our setup, and the application of global analysis, we were able to obtain these emission spectra "directly".…”

Section: Discussionmentioning

confidence: 99%

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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Fluorescence decay of tryptophan conformers in aqueous solution

Cited by 573 publications

References 12 publications

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

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

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Fluorescence decay of tryptophan conformers in aqueous solution

Cited by 573 publications

References 12 publications

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

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

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Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator