Steady-state fluorescence quenching applications for studying protein structure and dynamics

Mátyus, László; Szöllősi, János; Jenei, Attila

doi:10.1016/j.jphotobiol.2005.12.017

Cited by 147 publications

(101 citation statements)

References 76 publications

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Section: Effect Of Gst Pi Peptide Fragments On the Fluorescence Of 1-mentioning

confidence: 69%

“…4, spectrum a) with that of free tryptophan (Fig. 4, spectrum d), we observed a shift in the maximum emission peak likely due to shielding of the tryptophan residues in the proteins from the aqueous phase [20].A comparison of the emission spectrum (with excitation at 280 nm) of 2 μM of 1-Cys Prx alone (Fig. 4, spectrum a) with that of the same concentration of 1-Cys Prx in the presence of 3.4 μM GST pi peptide 41-85 (Fig.…”

mentioning

confidence: 70%

“…6), which enabled us to monitor the interaction between the GST peptides and 1-Cys Prx by means of the intrinsic fluorescence of these chromophores. These tryptophan residues of 1-Cys Prx exist in a solvent-restricted or hydrophobic environment as shown by the blue shift in the maximum emission peak observed with 1-Cys Prx when compared to free tryptophan [20]. In the presence of four GST pi peptides, a decrease in the maximum fluorescence of 1-Cys Prx at 330 nm is observed as compared to the 1-Cys Prx alone, which suggests that these fragments interact with 1-Cys Prx, further shielding it from the solvent or quenching the tryptophans [20].…”

Section: Author Manuscript Author Manuscriptmentioning

confidence: 99%

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Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Ralat¹,

Misquitta²,

Manevich³

et al. 2008

Archives of Biochemistry and Biophysics

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Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. KeywordsGlutathione S-transferase pi; 1-Cys peroxiredoxin; Heterodimer Glutathione S-transferases (GSTs 1 ), which catalyze the nucleophilic attack by the thiol of glutathione on electrophilic substrates, constitute a family of enzymes important in the detoxification of xenobiotics, endogenous compounds, and the products of oxidative stress [1,2]. The pi isozyme (GST pi), crystallized as a homodimer with a subunit molecular * Corresponding author. Fax: +1 302 831 6335. rfcolman@chem.udel.edu (R.F. Colman). 1 Abbreviations used: GST, glutathione S-transferase; GST pi, pi-class glutathione S-transferase; 1-Cys Prx, 1-Cysteine peroxiredoxin; Ni-NTA, nickel-nitriloacetic acid agarose; TRIS, tris (hydroxymethyl) aminomethane; EDTA, disodium ethylenediamine tetraacetate; MES, 2-(N-morpholino) ethane-sulfonate; WT, wild-type; GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript weight of 23,500, is of particular interest because it exhibits diverse roles in mammalian cells: it provides a defense against carcinogenesis, since it catalyzes the inactivation of known carcinogens [3]; it contributes significantly to the development of resistance to cancer chemotherapy, since GST pi levels increase in tumors and the enzyme metabolizes key anticancer drugs [4][5][6][7]; and it promotes the cellular response to oxidative stress, since GST pi has recently been reported to activate the anti-oxidant enzyme 1-Cys peroxiredoxin [8,9].1-Cys peroxiredoxin (1-Cys Prx, Prdx 6, Prx VI, and AOP2), a homodime...

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Section: Effect Of Gst Pi Peptide Fragments On the Fluorescence Of 1-mentioning

confidence: 69%

mentioning

confidence: 70%

Section: Author Manuscript Author Manuscriptmentioning

confidence: 99%

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Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Ralat¹,

Misquitta²,

Manevich³

et al. 2008

Archives of Biochemistry and Biophysics

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“…FRET measurements between Trp-237 and MantADP-Mg were performed with 15 M ⌬HPrK/P at exc ϭ 295 nm and em ϭ 440 nm at 600 V with a 5 nm band pass. For both FRET and MantADP experiments, the fluorescence of equivalent amounts of free MantADP in the absence of ⌬HPrK/P was recorded under the same conditions, and the linear regression coefficient of the corresponding curve was used for correction of the binding data (27).…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

Chaptal

Vincent

Gueguen-Chaignon

et al. 2007

Journal of Biological Chemistry

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The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phosphocarrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6 Å resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.In Gram-positive bacteria, HPr kinase/phosphorylase (HPrK/P) 6 is the central regulator of carbon catabolite repression, the paradigm of signal transduction (1). In response to certain metabolites that change their concentration in the presence of rapidly metabolizable carbon sources, HPrK/P either phosphorylates or dephosphorylates Ser-46 of the histidine-containing protein HPr. In Gram-positive bacteria, Ser(P)-46-HPr functions as co-repressor for carbon catabolite repression by interacting with the transcriptional regulator CcpA (catabolite control protein A) that regulates the expression of many genes (2, 3) (for a review, see Ref. 4). In the Gram-negative Neisseria meningitidis devoid of CcpA-mediated carbon catabolite repression, Ser(P)-46-HPr is involved in the cell adhesion process (5), suggesting that HPrK/P might constitute a potential target for new antimicrobial agents in pathogenic bacteria. Finally, the formation of Ser(P)-46-HPr inhibits PrfA, a transcription activator of several virulence genes in Listeria monocytogenes (6).HPrK/P was first identified as a serine protein kinase, and this activity is enhanced in the presence of glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and inhibited in the presence of inorganic phosphate (P i ) (7). Dephosphorylation of Ser(P)-46-HPr by HPrK/P was assumed to be hydrolytic. However, P i was also reported to stimulate the HPrK/P-c...

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“…In previous studies fluorescence quenching proved to be a powerful method to describe the conformational changes of macromolecules [21][22][23][24]. We applied acrylamide as a quencher and characterise the accessibility of the IAEDANS label attached to Cys 374 of the actin protomers.…”

Section: Introductionmentioning

confidence: 99%

The effects of formins on the conformation of subdomain 1 in actin filaments

Ujfalusi

Barkó

Hild

et al. 2010

Journal of Photochemistry and Photobiology B: Biology

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In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys 374 and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K SV ). Simultaneously the fraction of the fluorophore population accessible for the quencher (α) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.

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Steady-state fluorescence quenching applications for studying protein structure and dynamics

Cited by 147 publications

References 76 publications

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The effects of formins on the conformation of subdomain 1 in actin filaments

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