Optimization and Applications of Fluorescence anisotropy assays and Fluorescence Resonance Energy Transfer Measurements
Issue Date
2009-01-01Author
Liyanage, Mangala Roshan
Publisher
University of Kansas
Format
172 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Chemistry
Rights
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Metadata
Show full item recordAbstract
Calmodulin (CaM) is a calcium signaling protein that activates over hundred of targets including PMCA. This dissertation mainly focuses on optimizing and applications of fluorescence anisotropy (FA) and FRET experiments for CaM-target interactions. First we evaluated the extent of interaction of fluorophores with CaM upon conjugation. In this study, three dyes were tested for influences of their charges on interaction with CaM. We employed time-resolved and steady state fluoresce anisotropy as well as fluorescence quenching experiments to study these interactions. The positively charged dye turns out to strongly interact with CaM than neutral and negatively charged dyes. Secondly, FA based assays for direct determination of affinities of CaM-target interactions are developed and the results are consistent with previously reported values. Finally, a FRET based methods are used to study the mechanism of activation of PMCA by CaM and found that the results are consistent with previously reported three-state model.
Collections
- Chemistry Dissertations and Theses [335]
- Dissertations [4626]
Items in KU ScholarWorks are protected by copyright, with all rights reserved, unless otherwise indicated.
We want to hear from you! Please share your stories about how Open Access to this item benefits YOU.