Probing the Mechanism of Binding and Recognition of Methylated Lysine
Public Deposited- Last Modified
- March 19, 2019
- Creator
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Koenig, Amber
- Affiliation: College of Arts and Sciences, Department of Chemistry
- Abstract
- Lysine methylation is an important posttranslational modification that is responsible for the proper regulation of gene expression. The misregulation of these methylation marks has been linked to various diseases. Proteins that are involved in the regulation and recognition of these marks are emerging therapeutic targets. Detailed understanding of the mechanism employed by these proteins to recognize their natural substrates would provide valuable information for the development of probes with the necessary affinity and specificity required to provide activity and avoid off target effects. Cation-π interactions are thought to be one of the major noncovalent interactions contributing to methylated lysine recognition. Here we have demonstrate that two tyrosine residues present in the binding pocket of the reader protein heterochromatin protein 1 (HP1) show differential contributions to trimethyllysine recognition. By incorporating unnatural amino acids containing substitutions on the aromatic rings, we tune the ability of these residues to participate in cation-π interactions, which influences overall binding affinity. We demonstrate a clear linear free energy relationship (LFER) at both tyrosine positions of different magnitudes. In order to probe cation-π interactions with tryptophan mutations, we report synthetic methods for incorporation of unnatural amino acids that are not amenable to in vivo unnatural amino acid mutagenesis. We demonstrate the synthesis of Fmoc-protected fluorinated tryptophan for use in solid phase peptide synthesis, as well as an improved method for synthetically accessing long peptide or short protein sequences. By acetyl capping after coupling steps of solid phase peptide synthesis we have eliminated the possibility of deletion products arising from incomplete coupling reactions. By adding a polyhistidine tag at the N- terminus, we greatly simplified purification by incorporating an affinity tag that allows the isolation of only the fully synthesized protein. Furthermore, the function of the synthetic protein was confirmed by performing a binding assay with its native substrate. Lastly, we discuss ongoing efforts to expand studies to other reader proteins, as well as other substrates, including dimethyllysine, inhibitors, and a neutral analog. By studying other proteins that contain different binding pockets, such as lower methylation state readers that incorporate salt bridges, we can develop a broader and more complete understanding of the mechanism of recognition of these post-translational modifications. This information not only provides information for therapeutic design, but a fully characterized system for studying cation-π interactions can be a useful system for testing computational methods aimed at modeling these binding interactions for medicinal chemistry purposes.
- Date of publication
- August 2016
- Keyword
- DOI
- Resource type
- Rights statement
- In Copyright
- Advisor
- Brustad, Eric
- Waters, Marcey
- Meek, Simon
- Gagne, Michel
- Johnson, Jeffrey
- Degree
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill Graduate School
- Graduation year
- 2016
- Language
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