New Methods for Site-Specific Protein Labelling

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The human genome has been sequenced, allowing the identification of a huge number of putative genes. However, much work remains to be done to determine which genes are translated into proteins, where these proteins are located, and what biological roles they play, under different cellular conditions. To help address these critical fundamental questions, more powerful methods for monitoring the expression, localization and dynamic trafficking of specific proteins inside living cells are urgently required. Over the next funding period the Keillor research program will lead in an exciting new direction. We will address this urgent need by developing novel methods for the intracellular labelling of specific proteins, allowing their detection by fluorescence microscopy. The development of such methods will require a broad multidisciplinary approach that includes organic synthesis, physical organic studies and protein engineering. Our specific objectives comprise the development of two complementary approaches for protein labelling - one based on enzyme-mediated labelling, and the other relying on the selective fluorogenic reactivity of small molecules. In the enzyme–based approach, we will use protein engineering to develop a bacterial enzyme that can recognize a specific peptide sequence genetically fused to a protein of interest, expressed in a mammalian cell. The engineered enzyme will have the ability to incorporate small synthetic substrates into the target peptide tag sequence. In turn, these substrates will bear functional groups that allow for direct detection and imaging. Alternatively, the substrates may also be designed to undergo bioorthogonal reactions with other synthetic molecules that facilitate detection. In our complementary small molecule approach, labelling agents will be synthesised that become fluorescent upon their reaction with a different specific peptide tag, genetically encoded on the protein of interest. We will perform spectroscopic studies on these fluorogenic agents, to optimize the increase of their fluorescence upon their addition reaction. Kinetic studies will also be performed to guide the control of their reactivity, thereby ensuring the specificity of their fluorogenic addition reaction with the intended target peptide sequence, even in living cells. The breadth of our expertise heavily favours the feasibility of the proposed studies. Our repertoire of demonstrated methodology includes organic synthesis, kinetic and spectroscopic studies, molecular biology, protein expression, mammalian cell culture and fluorescence microscopy. Ultimately, we will apply our new labelling methods to the cell-based investigation of the conformation, mechanism and inhibition of enzymes such as the transglutaminases. We have studied the in vitro activity and inhibition of this class of enzymes for nearly 20 years; the new labelling techniques developed in the next phase of our research program will transform our powers of investigation, allowing us, and others, to pursue the study of enzymes on an entirely new, and highly relevant, cellular level.