Optimization and Applications of Fluorescence anisotropy assays and Fluorescence Resonance Energy Transfer Measurements

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.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2009

Author dc:creator

• Liyanage, Mangala Roshan

Advisor dc:contributor.advisor

• Johnson, Carey K

• Analytical chemistry
• Biophysics
• Calcium signaling
• Calmodulin
• Competitive assays
• Fluorescence anisotropy
• Fret
• Pmca

Statement dc:rights

• This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.

Language dc:language.iso

EN

Dc Identifier Other

http://dissertations.umi.com/ku:10327

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/5254