High-Sensitivity Fluorescence Anisotropy Detection of Protein-Folding Events: Application to α-Lactalbumin

Canet, Denis; Doering, Klaus; Dobson, Christopher M.; Dupont, Yves

doi:10.1016/s0006-3495(01)76169-3

Cited by 44 publications

(42 citation statements)

References 23 publications

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“…Stopped-flow fluorescence anisotropy was measured with a single photomultiplier and no polarizer in the emission light path. A similar set up has been reported recently in a study on protein folding in solution (23). In the anisotropy set-up the excitation light is modulated with a photoelastic modulator to give alternating horizontally and vertically polarized light with a frequency of 100 kHz.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Engel

Mierlo²,

Visser

2002

Journal of Biological Chemistry

103

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Conformational changes of bovine ␣-lactalbumin induced by adsorption on a hydrophobic interface are studied by fluorescence and circular dichroism spectroscopy. Adsorption of bovine ␣-lactalbumin on hydrophobic polystyrene nanospheres induces a non-native state of the protein, which is characterized by preserved secondary structure, lost tertiary structure, and release of calcium. This partially denatured state therefore resembles a molten globule state, which is an intermediate in the folding of bovine ␣-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k 1 ϳ 50 s ؊1 and k 2 ϳ 8 s ؊1 . The rate of partial unfolding is remarkably fast and even faster than unfolding induced by the addition of 5.4 M guanidinium hydrochloride to native ␣-lactalbumin. The large unfolding rates exclude the possibility that unfolding of bovine ␣-lactalbumin to the intermediate state occurs before adsorption takes place. Stoppedflow fluorescence anisotropy experiments show that adsorption of bovine ␣-lactalbumin on polystyrene nanospheres occurs within the dead time (15 ms) of the experiment. This shows that the kinetic processes as determined by stopped-flow fluorescence spectroscopy are not affected by diffusion or association processes but are solely caused by unfolding of bovine ␣-lactalbumin induced by adsorption on the polystyrene surface. A scheme is presented that incorporates the results obtained and describes the adsorption of bovine ␣-lactalbumin.Adsorption of proteins on solid/liquid interfaces is a generally occurring phenomenon both in nature and in man-made systems. It is well recognized that proteins undergo conformational changes upon adsorption on solid/liquid interfaces (1-3). Because unfolding of proteins can have a large effect on their function or properties, it is important to increase the knowledge about protein adsorption and about the resulting conformational changes. It is necessary to study not only the conformation of a protein in the adsorbed state but also the kinetics of the adsorption-induced conformational changes. This knowledge will help in controlling wanted or unwanted conformational changes of adsorbed proteins.In this work we focus on the kinetics of adsorption-induced conformational changes of bovine ␣-lactalbumin (BLA) 1 upon interaction with colloidal polystyrene nanospheres. Detailed information on adsorption-induced conformational changes and especially on the kinetics of adsorption-induced conformational changes is sparse. An important reason for this is the experimental difficulty of the presence of an adsorbent, which is usually a solid phase. A few routes have been designed to remove this difficulty. A macroscopic solid/water interface can be used in combination with a reflective technique like ellipsometry or total internal reflection fluorescence (4, 5). In other cases a microscopic system has been used that consists of small particles with diameters ranging from nanometers to micrometers in size (6, 7). The latter syst...

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Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Engel

Mierlo²,

Visser

2002

Journal of Biological Chemistry

103

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“…Measurements were taken of samples in a 1.0 ϫ 0.4-cm cuvette stirred continuously at the indicated temperature Ϯ 1°C. To maximize sensitivity, a single polarizer configuration was used to determine fluorescence anisotropy, A, and fluorescence intensity, Fl (V) (22). Fluorescence is reported as quenching, Q ϭ 1 Ϫ (Fl/Fl o ) where Fl o is the signal from oligonucleotide alone.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA

Defais

Phez

Johnson

2003

Journal of Biological Chemistry

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RecA protein from Escherichia coli catalyzes DNA strand exchange during homologous recombination in a reaction that requires nucleoside triphosphate cofactor. In the first step of this reaction RecA protein polymerizes on single-stranded DNA to form a filament with a stoichiometry of three nucleotides/RecA monomer called the presynaptic complex. We have used fluorescence anisotropy of a fluorescein-labeled oligonucleotide to investigate presynaptic complex formation. RecA-ATP␥S bound to oligonucleotide by a two-step process. Kinetic studies revealed an intermediate in the polymerization reaction that had greater mobility than the final product filament. The intermediate was transformed into the final product by a process that was independent of filament concentration and temperature, k ‫؍‬ 0.3 ؎ 0.1 min ؊1 . This process had the same rate as that reported for a step in the isomerization of presynaptic complex by ATP␥S (Paulus, B. F., and Bryant, Homologous recombination of DNA is a universal biochemical process required for chromosomal segregation and certain mechanisms of DNA replication and repair (1). Recombinases act in nucleoprotein filaments whose essential features have been conserved from phage to man (2, 3). The most thoroughly studied recombinase, Escherichia coli RecA protein, carries out the principle reactions of homologous recombination in vitro (2, 4). RecA protein has two functional DNA binding sites (5). In the first step of the reaction, multiple RecA monomers bind ssDNA 1 at one site of the protein to form a right-handed helical polymer, 3 nt per RecA monomer, known as a presynaptic complex. In the presence of ATP, this filament binds dsDNA, and if the DNA sequences are complementary, strand exchange occurs.RecA protein catalyzes DNA-dependent ATP hydrolysis uniformly throughout the nucleoprotein filament (6). The presynaptic complex is necessary and sufficient for ATPase activity, even though this filament can bind additional ssDNA or dsDNA (7). The effect of ATP hydrolysis on the DNA strand exchange reaction carried out by RecA protein has been investigated using stable ATP analogs such as ATP␥S (8, 9) or the mutant RecA protein K72R (10, 11). In these conditions the presynaptic filament binds but does not hydrolyze cofactor. This filament nevertheless carries out limited DNA strand exchange and is considered to reproduce some of the early steps of homologous recombination. In particular, binding of ATP or ATP␥S produces a filament in which DNA is extended (5.1 Å per base pair) and underwound (18 base pair/turn) (12-14). Concomitantly ATP or ATP␥S increase the stability of the nucleoprotein filament, while ADP destabilizes the filament relative to no cofactor (15).The mechanism by which RecA protein forms the presynaptic complex is poorly understood. Equilibrium binding studies have been carried out for the reaction of RecA with ssDNA in the absence of cofactor (15, 16). However, equilibrium constants and rate constants for formation of the presynaptic complex with ATP or ATP␥S have not been...

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“…26 As a consequence, in the case of κ-caseins, which contain tryptophan residues, it should be sensitive to oligomerization. Figure 6e shows the concentration dependence of fluorescence anisotropy of the unglycosylated and glycosylated form of SCM κ-casein.…”

Section: Fibrillation Process Of Scm κ-Casein As Analyzed By Tem and mentioning

confidence: 99%

Kinetics of Fibril Formation of Bovine κ-Casein Indicate a Conformational Rearrangement as a Critical Step in the Process

Léonil

Henry

Jouanneau

et al. 2008

Journal of Molecular Biology

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High-Sensitivity Fluorescence Anisotropy Detection of Protein-Folding Events: Application to α-Lactalbumin

Cited by 44 publications

References 23 publications

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA

Kinetics of Fibril Formation of Bovine κ-Casein Indicate a Conformational Rearrangement as a Critical Step in the Process

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