Full metadata
Title
Isolation and functional studies of the F-type ATP synthase from spinach chloroplasts and Heliobacterium modesticaldum
Description
Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP synthesis is coupled to proton translocation is not known as no structure of the intact ATP-synthase nor the intact FO subcomplex has been determined to date. Structural information may shed light on these mechanisms and aid in understanding how structural changed relate to its coupling to ATP synthesis. The work in this thesis has successful established a defined large-scale CF1FO isolation procedure resulting in high purity and high yield of this complex from spinach thylakoid membranes by incorporating a unique combination of biochemical methods will form the basis for the subsequent structural determination of this complex. Isolation began from the isolation of intact chloroplasts and the separation of intact thylakoid membranes. Both native and denaturing electrophoresis analyses clearly demonstrated that the purified CF1FO retains its quaternary structure consisting of the CF1 and CFO subcomplexes and nine subunits (five F1 subunits: α, β, γ, δ and ε, and four FO subunits: a, b, b' and c). Moreover, both ATP synthesis and hydrolysis activities were successfully detected using protein reconstitution in combination with acid-base incubation and in-gel ATPase assays, respectively. Furthermore, the ATP-synthase of H. modesticaldum, an anaerobic photosynthetic bacterium, was also isolated and characterized at the biochemical level. These biochemical characterizations directly influenced recent studies on the high-resolution structure determination of intact CF1FO using electron crystallography on two-dimensional crystals. The availability of the functionally intact CF1FO purified at a large scale will lead to studies that investigate the possible crystallization conditions to ultimately determine its three-dimensional structure at atomic resolution.
Date Created
2015
Contributors
- Yang, Jay-How (Author)
- Fromme, Petra (Thesis advisor)
- Redding, Kevin (Committee member)
- Gould, Ian (Committee member)
- Arizona State University (Publisher)
Topical Subject
Resource Type
Extent
xv, 203 pages : illustrations (mostly color)
Language
Copyright Statement
In Copyright
Primary Member of
Peer-reviewed
No
Open Access
No
Handle
https://hdl.handle.net/2286/R.I.29992
Statement of Responsibility
by Jay-How Yang
Description Source
Retrieved on Aug. 18, 2015
Level of coding
full
Note
Partial requirement for: Ph.D., Arizona State University, 2015
Note type
thesis
Includes bibliographical references (pages 168-188)
Note type
bibliography
Field of study: Biochemistry
System Created
- 2015-06-01 08:17:33
System Modified
- 2021-08-30 01:28:28
- 2 years 7 months ago
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