Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins in living cells, allowing the elucidation of their localization, trafficking and ultimately their cellular function. We have developed a novel labelling technique based on the genetic fusion of a protein of interest to a small helical peptide sequence containing two Cys residues (dC10). This tag can undergo an efficient reaction with small fluorogenic labelling agents composed of a fluorophore and a dimaleimide core (dM10) that confers high reaction specificity, and quenches the latent fluorescence through photo-induced electron transfer, until both of its maleimide groups have formed robust covalent bonds with the tag Cys thiol groups.
Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivity of the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, we re-engineered the dC10 tag through rational protein design. Mutant libraries were prepared through combinatorial mutation at specific positions of the helical tag sequence, and screened for their fluorogenic reactivity. In this way, we identified a novel sequence for a next-generation dC10 tag that confers 10-fold greater selectivity that we then applied to in cellulo labelling. Subsequent mechanistic studies revealed the basis for this dramatic increase in reactivity.
Current applications of this powerful labelling technique, including the site-specific chelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labelling for X-ray crystallography, will also be discussed.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/34175 |
| Date | January 2016 |
| Creators | Strmiskova, Miroslava |
| Contributors | Keillor, Jeffrey W, Goto, Natalie |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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