The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main liga... more The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation be...
During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vin... more During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ∼4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.
... Antonie JWG Visser,* Kees Vos, Arie van Hoek,t and Jillert S. Santema Department of Biochemis... more ... Antonie JWG Visser,* Kees Vos, Arie van Hoek,t and Jillert S. Santema Department of Biochemistry, Agricultural University, 6703 BC Wageningen, The Netherlands (Received: April 2, 1987: In Final Form ... Rhodamine B and rose bengal were obtained from Eastman Kodak. ...
The time-resolved and steady-state fluorescence techniques were employed to elucidate possible in... more The time-resolved and steady-state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4-naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water-ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision-interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase.
Fluorescent base analogues such as 2-aminopurine report DNA dynamics on the scale of single bases... more Fluorescent base analogues such as 2-aminopurine report DNA dynamics on the scale of single bases. We find that the time-dependent fluorescence of various 2-aminopurine-containing dinucleotides can be described by only two components: a fast (∼20ps) exponential decay and a much slower (∼1ns) stretched exponential. This is much simpler than previously proposed models. The fast component reflects quenching in the stacked
The two ß-cyclodextrin-calix[4]arene couples 1 and 2 were prepared as sensing molecules for the d... more The two ß-cyclodextrin-calix[4]arene couples 1 and 2 were prepared as sensing molecules for the detection of organic analytes in water. Compounds 1 and 2 are amphiphilic in nature and form aggregates in aqueous solution. Compound 1 forms vesicles both in the absence and in the presence of guest species, and its fluorescence intensity does not change. Compound 2 forms fibers, which change into vesicles upon guest addition. This behavior is accompanied by a reduction in fluorescence intensity. The aggregates were visualized by transmission electron microscopy using both the freeze fracture technique and the uranyl staining method. Langmuir monolayer experiments show that intermolecular interactions lead to a preorganization of 2, whereas molecules of 1 behave independently analogous to conventional amphiphiles. Fluorescence anisotropy decay measurements give evidence for rapid internal dye motion in the aggregates of both compounds 1 and 2. In addition, a slower decay process of low amplitude is observed for both compounds, indicating free rotational motion of single molecules of 1 but the absence of rotational motion of individual molecules within the aggregates of 2. This difference indicates the intermolecular complexation of the fluorophores in the aggregates of 2. The fluorescence lifetimes of aqueous solutions of 2 reveal that the reduction in fluorescence intensity is based on static quenching by the amino group present in the spacer of 2. Our results show the presence of vesicular bilayers of independent amphiphiles for 1, and for 2 the formation of assemblies of molecular threads which are composed by interconnective, linear host-guest complexation.
Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent protein... more Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent
The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main liga... more The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation be...
During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vin... more During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ∼4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.
... Antonie JWG Visser,* Kees Vos, Arie van Hoek,t and Jillert S. Santema Department of Biochemis... more ... Antonie JWG Visser,* Kees Vos, Arie van Hoek,t and Jillert S. Santema Department of Biochemistry, Agricultural University, 6703 BC Wageningen, The Netherlands (Received: April 2, 1987: In Final Form ... Rhodamine B and rose bengal were obtained from Eastman Kodak. ...
The time-resolved and steady-state fluorescence techniques were employed to elucidate possible in... more The time-resolved and steady-state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4-naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water-ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision-interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase.
Fluorescent base analogues such as 2-aminopurine report DNA dynamics on the scale of single bases... more Fluorescent base analogues such as 2-aminopurine report DNA dynamics on the scale of single bases. We find that the time-dependent fluorescence of various 2-aminopurine-containing dinucleotides can be described by only two components: a fast (∼20ps) exponential decay and a much slower (∼1ns) stretched exponential. This is much simpler than previously proposed models. The fast component reflects quenching in the stacked
The two ß-cyclodextrin-calix[4]arene couples 1 and 2 were prepared as sensing molecules for the d... more The two ß-cyclodextrin-calix[4]arene couples 1 and 2 were prepared as sensing molecules for the detection of organic analytes in water. Compounds 1 and 2 are amphiphilic in nature and form aggregates in aqueous solution. Compound 1 forms vesicles both in the absence and in the presence of guest species, and its fluorescence intensity does not change. Compound 2 forms fibers, which change into vesicles upon guest addition. This behavior is accompanied by a reduction in fluorescence intensity. The aggregates were visualized by transmission electron microscopy using both the freeze fracture technique and the uranyl staining method. Langmuir monolayer experiments show that intermolecular interactions lead to a preorganization of 2, whereas molecules of 1 behave independently analogous to conventional amphiphiles. Fluorescence anisotropy decay measurements give evidence for rapid internal dye motion in the aggregates of both compounds 1 and 2. In addition, a slower decay process of low amplitude is observed for both compounds, indicating free rotational motion of single molecules of 1 but the absence of rotational motion of individual molecules within the aggregates of 2. This difference indicates the intermolecular complexation of the fluorophores in the aggregates of 2. The fluorescence lifetimes of aqueous solutions of 2 reveal that the reduction in fluorescence intensity is based on static quenching by the amino group present in the spacer of 2. Our results show the presence of vesicular bilayers of independent amphiphiles for 1, and for 2 the formation of assemblies of molecular threads which are composed by interconnective, linear host-guest complexation.
Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent protein... more Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent
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