ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
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- Creators: Redding, Kevin
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.
ContributorsYang, Jay-How (Author) / Fromme, Petra (Thesis advisor) / Redding, Kevin (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2015
Description
Biomolecules can easily recognize its corresponding partner and get bound to it, resulting in controlling various processes (immune system, inter or intracellular signaling) in biology and physiology. Bonding between two partners can be a result of electrostatic, hydrophobic interactions or shape complementarity. It is of great importance to study these kinds of biomolecular interactions to have a detailed knowledge of above mentioned physiological processes. These studies can also open avenues for other aspects of science such as drug development. Discussed in the first part of Chapter 1 are the biotin-streptavidin biomolecular interaction studies by atomic force microscopy (AFM) and surface plasmon resonance (SPR) instrument. Also, the basic working principle of AFM and SPR has been discussed.
The second part of Chapter 1 is discussed about site-specific chemical modification of peptides and proteins. Proteins have been used to generate therapeutic materials, proteins-based biomaterials. To achieve all these properties in protein there is a need for site-specific protein modification.
To be able to successfully monitor biomolecular interaction using AFM there is a need for organic linker molecule which helps one of the investigating molecules to get attached to the AFM tip. Most of the linker molecules available are capable of investigating one type of interaction at a time. Therefore, it is significant to have linker molecule which can monitor multiple interactions (same or different type) at the same time. Further, these linker molecules are modified so that biomolecular interactions can also be monitored using SPR instrument. Described in Chapter 2 are the synthesis of organic linker molecules and their use to study biomolecular interaction through AFM and SPR.
In Chapter 3, N-terminal chemical modification of peptides and proteins has been discussed. Further, modified peptides are attached to DNA thread for their translocation through the solid-state nanopore to identify them. Synthesis of various peptide-DNA conjugates and their nanopore studies have been discussed in this chapter.
The second part of Chapter 1 is discussed about site-specific chemical modification of peptides and proteins. Proteins have been used to generate therapeutic materials, proteins-based biomaterials. To achieve all these properties in protein there is a need for site-specific protein modification.
To be able to successfully monitor biomolecular interaction using AFM there is a need for organic linker molecule which helps one of the investigating molecules to get attached to the AFM tip. Most of the linker molecules available are capable of investigating one type of interaction at a time. Therefore, it is significant to have linker molecule which can monitor multiple interactions (same or different type) at the same time. Further, these linker molecules are modified so that biomolecular interactions can also be monitored using SPR instrument. Described in Chapter 2 are the synthesis of organic linker molecules and their use to study biomolecular interaction through AFM and SPR.
In Chapter 3, N-terminal chemical modification of peptides and proteins has been discussed. Further, modified peptides are attached to DNA thread for their translocation through the solid-state nanopore to identify them. Synthesis of various peptide-DNA conjugates and their nanopore studies have been discussed in this chapter.
ContributorsBiswas, Sudipta (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Redding, Kevin (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016