Thesis
Quantized "off-on" reversible fluorescent molecular switch for phosphatidylserine
Washington State University
Master of Science (MS), Washington State University
2010
Handle:
https://hdl.handle.net/2376/103794
Abstract
Phosphatidylserine lipid is a critical constituent of cell membranes and an imbalance in its expression level is an indicator for ailments like cancer and Alzheimer's disease. Yet a robust technology for detecting and measuring it in vivo and in vitro does not exist. The biosensor Lact-C2-NBD that consists of two NBD (nitrobenzodioxazole) molecules attached to proximal cysteine residues on the C2 domain of the protein lactadherin (Lact-C2) developed in this study is a powerful biocompatible protein fluorescent molecule that addresses this technological need. It has been shown that the red fluorescence (670 nm emission) of proximal NBD molecules in Lact-C2-NBD probe shifts to green fluorescence (540 nm emission) upon binding to phosphatidylserine in the outer membrane of model phosphatidylserine/phosphatidylcholine vesicles due to the proximal to distal movement of the NBD molecules. The red fluorescence is restored when the probe unbinds and migrates through the lipid bilayer when the NBD molecules are again proximal. The red to green fluorescence shift is observed again when the probe binds to phosphatidylserine in the inner membrane. This entire process could be monitored by spectrofluorimetry and fluorescence microscopy in model vesicles and normal and cancer prostate cells. The fluorescence shift is likely due to the coupling of the electronic energy levels of NBD molecules when they are proximal (red fluorescence) and decoupling of this interaction when they are distal. This proximal to distal movement of the NBD molecules is caused by the binding of Lact-C2-NBD to phosphatidylserine and it is both photoaccelarated and reversible. The Lact-C2-NBD phosphatidylserine probe is photostable and membrane permeable and as a result can target this lipid both in the outer and inner membranes of vesicles and cells.
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Details
- Title
- Quantized "off-on" reversible fluorescent molecular switch for phosphatidylserine
- Creators
- Shailin Kamlesh Patel
- Contributors
- Subra Muralidharan (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525045701842
- Language
- English
- Resource Type
- Thesis