Conformational Changes of Chloroplast ATP Synthase γ Subunit Studied by Single Molecule Detection Methods
Date Issued
2007
Date
2007
Author(s)
Wu, Shang-Jhen
DOI
zh-TW
Abstract
The chloroplast F0F1-ATP synthase - ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the subunit of the chloroplast F1 enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthase of higher plants. Molecular dynamic studies predicted that the dithiol domain exists in a range of conformations between two extreme states；the fully open state, with the dithiol reduced ,and a fully closed state with dithiol oxidized to form a disulfide bridge. The open conformation is predicted to represent the activated state and the closed conformation is inactivated state. We isolated CF0F1 from spinach leaves. The γ subunit of spinach CF1 contains four cysteinyl sulfydryls, two of which (Cys199 and Cys205) are linked together to form the disulfide bridge. We isolated γ subunit from spinach ATPase by Hydroxyapatite Columns, and confirmed the native state of γ subunit by CD spectroscopy. We labeled TMR and Cy5 as donor and acceptor on γ subunit, confirm the numbers of dyes on γ subunit by ESI-MS, and confirmed the positions of dye on γ subunit by MALDI-TOF MS. Finally, we observed the change of conformations of γ subunit between folding and unfolding state by single molecular detection methods.
Subjects
單分子螢光偵測技術
Chloroplast ATP Synthase，CF0F1，γ subunit，dithiol containing domain
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-96-R94223035-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):8de479fa6c0ca9bc9cc08043eb19e5d1