The ATP synthase from spinach chloroplasts (Spinacea oleracea) : improved isolation and purification of the enzyme and conformational changes observed with fluorescent labels

The ATP Synthase from Chloroplasts, CF0F1, is an integral membrane enzyme, which catalyses the synthesis of ATP from ADP and Phosphate. The reaction is driven energetically by proton-transport across the enzyme. The activity of the enzyme is regulated through a transmembrane pH-difference and a redox process, in which a disulfide bridge in the g-subunit is reduced. The protein undergoes a conformational change during catalysis which can be investigated with the help of Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetimes (FL) or with Fluorescence Correlation Spectroscopy (FCS). <br>Fluorescent probes were used to selectively label different subunits of CF1, in order to conduct spectroscopic investigations of conformational changes which occur upon substrate binding to CF1. Results from FRET experiments with CF1 show that the distance between the two labelled sites g-Cys199(205) and a-Lys278 was changed upon addition of the non-hydrolyzable substrate AMP-PNP. <br> Fluorescence lifetime measurements of free and enzyme bound fluorophores confirm this effect. The fluorophores LY and Alexa 546 exhibited biphasic decays when bound to CF1 and free in solution. In the case of Alexa 546, the decay time of the faster component was 900 ps and was not changed upon binding to CF1. With LY the the decay time of the faster component was reduced from 120 ps to 70 ps upon binding to CF1. When CF1 was labelled with both fluorophores, the the decay time of the faster component was further reduced to 50 ps. This was accredited to FRET from LY to Alexa 546. <br> The translational diffusions coefficient of CF1 labelled with Alexa 546 was measured on single molecules using FCS. The translational diffusions coefficient was increased in the presence of AMP-PNP. This was interpreted as a reduction in volume of the enzyme upon substrate binding and demonstrated that conformational changes of CF1 could also be investigated using fluorescently labelled single molecules.

Author dc:creator

• Kirch, Robert Dale

Contributors dc:contributor

• Gräber, Peter

• Konformationsänderung
• Fluoreszenz Resonanz Energie Transfer (FRET)
• Fluoreszenzlebensdauer
• Einzelmolekülfluoreszenzspektroskopie
• ATP Synthese
• Enzyme Purification
• Conformational Change
• Fluorescence Resonance Energy Transfer (FRET)
• Fluorescence Lifetime
• Single Molecule Spectroscopy

Repository record source_url

https://freidok.uni-freiburg.de/data/666

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:666