Application of Small Molecule Receptors to the Analysis of Post-translational Modifications Using Fluorescent Indicator Displacement Assays.
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Peacor, Brendan. Application of Small Molecule Receptors to the Analysis of Post-translational Modifications Using Fluorescent Indicator Displacement Assays. Chapel Hill, NC: University of North Carolina at Chapel Hill Graduate School, 2015. https://doi.org/10.17615/qp86-ta72APA
Peacor, B. (2015). Application of Small Molecule Receptors to the Analysis of Post-translational Modifications Using Fluorescent Indicator Displacement Assays. Chapel Hill, NC: University of North Carolina at Chapel Hill Graduate School. https://doi.org/10.17615/qp86-ta72Chicago
Peacor, Brendan. 2015. Application of Small Molecule Receptors to the Analysis of Post-Translational Modifications Using Fluorescent Indicator Displacement Assays. Chapel Hill, NC: University of North Carolina at Chapel Hill Graduate School. https://doi.org/10.17615/qp86-ta72- Last Modified
- March 19, 2019
- Creator
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Peacor, Brendan
- Affiliation: College of Arts and Sciences, Department of Chemistry
- Abstract
- This dissertation focuses on the application of small molecule receptors to a fluorescent assay for the study of histone post-translational modifications, both in real-time enzyme reactions and endpoint characterization of analytes. In the first section, dynamic combinatorial chemistry was used to generate a series of A2X receptors that varied the functionality of the X monomer. This allowed us to systematically study the contribution of pocket depth and electrostatic interactions on binding methylated lysine. We discovered that changing the location of a carboxylate increased affinity to K(Me)2, presumably through a salt bridge, while an additional carboxylate increased affinity across the entire lysine series. Additionally, formation of a deeper binding pocket saw a selectivity increase for K(Me)3 over the lower methylation states. The remaining sections describe the application of the Waters lab suite of receptors to fluorescence indicator displacement assays (IDAs). In these assays, fluorescence signal is directly proportional to the competitive binding of a histone analyte. We applied a sensor system using the receptor A2N and the fluorophore Lucigenin (LCG) to study the enzymatic dimethylation of histone H3 lysine 9 by the methyltransferase G9a. Optimization of the enzymatic buffer system established an effective methyltransferase reaction to short histone 3 peptide substrates. Applying these conditions to the fluorescent assay we are able to monitor enzymatic activity, allowing future experiments to test enzyme response to neighboring modifications in the ‘histone code’. This assay was also applied to the preliminary examination of the arginine methyltransferases, demonstrating the general applicability of the assay to the full range of enzymatic methylation reactions. With the large number of receptors previously established, we sought to develop a general discriminatory assay capable of recognizing histone modifications beyond the designed scope of the sensor. By combining the fluorescent IDA signal for four different receptors, A2B, A2D, A2N, and A2G, we were able to accurately classify thirteen different histone peptides in a single output. Each peptide had multiple modifications, including arginine methylation and lysine methylation, as well as lysine methylation and threonine phosphorylation. The classification assay was able to distinguish both the degree of modification as well as the site of the specific modifications, all based on the slight perturbations neighboring residues make on binding affinity. This assay was also preliminarily applied to the sensing of complex enzymatic reactions by performing a mock kinase experiment, in which we were able to classify distinct ‘time-point’ of enzymatic phosphorylation on two separate substrates. The final section focuses on the expansion of the target class of analytes for the combinatorial sensor array. While the previous study focused on modification-neighboring methylation events, here we describe the classification of the neutral modifications of arginine and lysine, reactions that abolish positive charge and weaken the affinity of the analytes to the receptor. Notably we are able to identify peptides based not just on how many charges are lost, but which specific residues are neutralized and where in the sequence the modification takes place. This opens the door to a large number of enzymatic reactions and histone analytes for which traditional methods of study are insufficient.
- Date of publication
- August 2015
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- Rights statement
- In Copyright
- Advisor
- Waters, Marcey
- Johnson, Jeffrey
- Frye, Stephen
- Lawrence, David
- Weeks, Kevin
- Degree
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill Graduate School
- Graduation year
- 2015
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- Place of publication
- Chapel Hill, NC
- Access right
- There are no restrictions to this item.
- Date uploaded
- August 25, 2015
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