Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. Sylvia Daunert

Abstract

The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. The bioluminescent protein, aequorin, has been employed as a reporter for decades due to its simplistic, non-hazardous nature and its high sensitivity of detection. More recently aequorin has been subject to spectral tuning. Techniques such as random and site-directed mutagenesis, the incorporation of coelenterazine analogues and the incorporation of non-natural amino acids have expanded the palette of aequorin by altering their emission wavelengths and/or half-lifes. Due to the increased diversity of aequorin, it can be used in multianalyte detection.

Although aequorin has been studied extensively and has been used as a reporter in a wide array of applications, it has never been employed as a reporter in systems that involve the splitting of aequorin. Herein we describe the splitting of aequorin in such a way where it becomes the reporter protein in the development of protein-based molecular switches. We have created two distinct protein switches by genetically inserting the glucose-binding protein and the sulfate-binding protein into the aequorin sequence, splitting it in such a manner that it allows for the selective detection of glucose and sulfate, respectively. In a separate investigation, we developed a bioluminescence inhibition binding assay for the detection of hydroxylated polychlorinated biphenyls. These systems have shown that they can be employed in the detection of the respective analyte in biological as well as in environmental samples, which demonstrated a sensitive, fast alternative approach to current methods for on-site screening.

Furthermore, we propose the rational design, preparation and use of truncated aequorin fragments in bioanalytical platforms such as multi-analyte detection, protein complementation assays and protein tagging assays based on our discovery that truncated aequorin retains partial bioluminescence emission. One such truncated aequorin demonstrated a large red shift in the emission maximum. It is envisioned that this new red-shifted truncated aequorin will find applications in multi-analyte detection. We anticipate that this work will lead to the discovery of additional functional truncated aequorin fragments that can be employed in novel protein-protein interactions or protein folding systems.

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