Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers

Valpuesta, José M.; Goñi, Félix M.; Macarulla, J. M.

doi:10.1016/0003-9861(87)90568-6

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…Protein intrinsic fluorescence spectra were recorded on a Perkin-Elmer LS 50B spectrofluorimeter with a temperature-controlled sample holder. The excitation wavelength used was 295 nm to minimize tyrosine emission (17). Chemical stability was measured after incubating HlyA or ProHlyA (10 µg/mL) at various concentrations (0-5 M) of GdnHCl buffered with TC buffer at 25 °C for 2 h. After incubation, the Trp fluorescence spectra were recorded in the range 320-400 nm using 150-µL cuvettes.…”

Section: Methodsmentioning

confidence: 99%

Fatty Acids Covalently Bound to α-Hemolysin of Escherichia coli Are Involved in the Molten Globule Conformation: Implication of Disordered Regions in Binding Promiscuity

Herlax

Bakás

2007

Biochemistry

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α-Hemolysin (HlyA) is a pore-forming toxin secreted by pathogenic strains of Escherichia coli. The toxin is synthesized as a protoxin, ProHlyA, which is matured in the cytosol to the active form by acylation at two internal lysines, K563 and K689 (HlyA). It is widely known that the presence of fatty acids is crucial for the hemolytic and cytotoxic effects of the toxin. However, no detailed physicochemical characterization of the structural changes produced by fatty acids in the soluble protein prior to membrane binding has been carried out to date. The effects of chemical denaturants, the ANS binding parameters (K d and n) and the sensitivity to proteases were compared between the acylated and unacylated protein forms HlyA and ProHlyA. Our results are consistent with a molten globular form of the acylated protein. Moreover, because molten globule proteins are intrinsically disordered proteins, using disorder prediction analyses, we show that HlyA contains 9 regions composed of 10−30 natively disordered amino acids. We propose that this conformation induced by covalently bound fatty acids might provide HlyA with the ability to bind to a variety of molecules during its action mechanism.

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Section: Methodsmentioning

confidence: 99%

Fatty Acids Covalently Bound to α-Hemolysin of Escherichia coli Are Involved in the Molten Globule Conformation: Implication of Disordered Regions in Binding Promiscuity

Herlax

Bakás

2007

Biochemistry

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“…The excitation wavelength used was 295 nm to minimize tyrosine emission. 14 Fluorescence was measured after incubating apoAI with LPS. Spectra were recorded in the range 320-400 nm using 150-ml cuvettes (4 nm slit width for both excitation and emission).…”

Section: Fluorescence Spectroscopymentioning

confidence: 99%

Contribution of the C-terminal end of apolipoprotein AI to neutralization of lipopolysaccharide endotoxic effect

2010

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It is well known that high density lipoprotein (HDL) binds bacterial lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of this work was to study changes in the apolipoprotein (apo) AI structure after its interaction with LPS as well as to determine the protein domain involved in that interaction. The presented data indicate that LPS does not lead to major changes in the structure of apoAI, judging from Trp fluorescence spectra. However, analysis of denaturation behavior and binding of ANS show that LPS induces a loosened protein conformation. Further evidence for an apoAI-LPS specific interaction was obtained by incubation of the protein with (125)I-ASD-LPS. The results show that multiple regions of the protein were able to interact with LPS, according to its amphiphatic nature. Finally, the contribution of the purified C-terminal fragment of the protein in the endotoxin neutralization was evaluated in comparison with the effect of apoAI. In both cases, the same decrease in tumor necrosis factor-α released was observed. This result suggests that the C-terminal half of apoAI is the main domain responsible of the neutralization effect of this protein. Our data may provide innovative pharmacological tools in endotoxin neutralization therapies.

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“…Protein intrinsic fluorescence spectra were recorded on a spectrofluorometer (SLM 4800 Aminco, Urbana, IL, USA) at a 0.1 mg/mL protein concentration in 100 mM sodium phosphate buffer, pH 7.5 using 1‐mL capacity quartz cuvettes. The chosen excitation wavelength was 295 nm to minimize tyrosine emission (Valpuesta et al, ). Slit width was 4 nm for both excitation and emission.…”

Section: Methodsmentioning

confidence: 99%

Characterization of phenoloxidase activity from spiderPolybetes pythagoricushemocyanin

et al. 2015

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Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygen transporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. In the present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricus hemocyanin (PpHc) PO activity to be 0.407 mM and 0.081 µmolmin(-1) mg protein(-1) , respectively. Dopamine was used as the substrate with SDS at a final concentration of 10 mM and a 30-min incubation at 25°C. Conformational changes in Hc associated with the SDS treatment were analyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. The secondary and tertiary structural changes of PpHc induced by SDS led to increases in α-helical content and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nm in the presence of SDS was also observed. These results suggest that the SDS-induced PO activity of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.

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Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers

Cited by 7 publications

References 35 publications

Fatty Acids Covalently Bound to α-Hemolysin of Escherichia coli Are Involved in the Molten Globule Conformation: Implication of Disordered Regions in Binding Promiscuity

Fatty Acids Covalently Bound to α-Hemolysin of Escherichia coli Are Involved in the Molten Globule Conformation: Implication of Disordered Regions in Binding Promiscuity

Contribution of the C-terminal end of apolipoprotein AI to neutralization of lipopolysaccharide endotoxic effect

Characterization of phenoloxidase activity from spiderPolybetes pythagoricushemocyanin

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