Dielectric relaxation in a single tryptophan protein.
TL;DR: A remarkable blue shift in fluorescence upon bimolecular quenching in the single‐tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity is reported.
About: This article is published in FEBS Letters.The article was published on 2001-12-07 and is currently open access. It has received 11 citations till now. The article focuses on the topics: Quenching (fluorescence) & Fluorescence.
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TL;DR: This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.
87 citations
01 Jan 2005
TL;DR: Red edge excitation shift (REES) as discussed by the authors is a phenomenon that occurs when the solvent shell around a polar fluorophore relaxes and shifts toward the red edge of the absorption band.
Abstract: 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
57 citations
Cites methods from "Dielectric relaxation in a single t..."
...79 Interestingly, the presence of slow solvent relaxation at ambient temperature has been demonstrated utilizing REES of the single tryptophan protein Bj2S, a seed albumin from Brassica juncea.(80) The REES approach has been used in a number of cases to monitor the tryptophan environment and dynamics...
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TL;DR: A novel trypsin inhibitor from Indian mustard Brassica juncea that is unique in being the precursor of a 2S seed storage protein that can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal.
53 citations
TL;DR: There is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum, and the absorption of GFP is primarily determined by intrinsic chromophore properties.
52 citations
Cites background from "Dielectric relaxation in a single t..."
...Such time-dependent fluorescence shifts have been observed in other protein environments (19,20)....
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TL;DR: In silico alignment of sequences of napin has revealed that the internal repeats spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species, which may contribute to the greater stability of nap in nature.
24 citations
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TL;DR: The spectral shifts and lifetime data indicate that red-edge excitation results in the selective excitation of "solvent-relaxed" fluorophores, and comparison of the data obtained for TNS in solvents and bound to the macromolecules may estimate the relaxation rate of the environment.
Abstract: In moderately polar and viscous solvents, the emission spectra of fluorophores often shift to longer wavelengths as the excitation wavelength is increased toward the long-wavelength (red) side of the absorption. Red shifts occur because long-wavelength excitation results in photoselection of those fluorophores which are interacting most strongly with the polar solvent molecules. The observation of excitation red shifts requires that these enhanced dipole-dipole interactions are maintained in the photoselected population during the lifetime of the excited state. Consequently, the magnitude of the excitation red shifts depends upon the dynamic properties of the environment surrounding the fluorophore, as well as upon the solvent polarity and the sensitivity of the fluorophore to the polarity of the solvent. We used this phenomenon to investigate the dynamic properties of reference solvents, model membranes, and the protein apomyoglobin labeled with 6-(p-toluidinyl)-2-naphthalenesulfonic acid (TNS). The spectral shifts and lifetime data indicate that red-edge excitation results in the selective excitation of "solvent-relaxed" fluorophores. By comparison of the data obtained for TNS in solvents and bound to the macromolecules, one may estimate the relaxation rate of the environment. This comparison indicates rapid spectral relaxation of TNS bound to lipid vesicles and a somewhat slower relaxation around TNS bound to the heme site of apomyoglobin.
142 citations
TL;DR: The determined structure of napin BnIb is compatible with activities attributed to these proteins such as phospholipid vesicle interaction, allergenicity, and calmodulin antagonism and will provide a firm basis for a rational use of genetic engineering in order to develop improved transgenic plants.
Abstract: Napin BnIb is a representative member of the 2S albumin seed proteins, which consists of two polypeptide chains of 38 and 84 kDa linked by two disulfide bridges In this work, a complete assignme
102 citations
78 citations
TL;DR: It is found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm, and their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation and emission wavelengths, indicating that the averagetryptophan environment in tubulin is motionally restricted.
Abstract: The fluorescence emission maximum of a polar fluorophore in viscous medium often shows a dependence on excitation wavelength, a phenomenon which is named red edge excitation shift (REES). We have found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm. Also, their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation and emission wavelengths. These results indicate that the average tryptophan environment in tubulin is motionally restricted. Although the tryptophan(s) responsible for the observed REES effect could not be localized, it could be concluded from energy transfer experiments with the tubulin-colchicine complex that the tryptophan(s) participating in energy transfer with bound colchicine probably does not contribute to the REES. A REES of 7 nm was also observed in the case of colchicine complexed with tubulin. However, such a REES was not seen in similar studies with the B-ring analogs of colchicine, viz. 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (called AC because it lacks the B ring of colchicine) and deacetamidocolchicine (which lacks the acetamido substituent at the C-7 position of the B ring). There may be two possible reasons to explain these data. (1) Structural differences between colchicine and its analogs may give rise to differences in their excited state dipole moments which will directly affect the extent of REES, and (2) The B-ring substituent, hanging outside the colchicine binding site on the beta-subunit of the tubulin dimer, probably makes contact with the alpha-subunit of tubulin and imparts a rigidity to that region of the protein, which facilitates the REES.
55 citations
TL;DR: It is shown that the fluorescence of multitryptophan-containing proteins, such as horse-liver alcohol dehydrogenase, 3-phosphoglycerate kinase and lysozyme, can be resolved for different segmental contributions, each characterized by collisional (Ki) and static (Vi) quenching constants.
Abstract: From acrylamide quenching results, analyzed by an itterative non-linear least-squares method, we have shown that the fluorescence of multitryptophan-containing proteins, such as horse-liver alcohol dehydrogenase, 3-phosphoglycerate kinase and lysozyme, can be resolved for different segmental contributions, each characterized by collisional (Ki) and static (Vi) quenching constants. The ability to resolve the heterogeneous fluorescence of proteins makes it possible to follow changes in dynamics of the individual residues. In yeast 3-phosphoglycerate kinase, which contains only two tryptophan residues, three fluorescent fractions, characterized by different accessibility to the quencher, were observed. Two of them are assigned to one of the tryptophan residue. This may be interpreted in terms of conformational fluctuations, which facilitate the access of acrylamide molecules to the buried tryptophan residues.
46 citations