Harnessing the flexibility of tubulin tyrosine ligase to site-specifically label C-terminus of alpha-tubulin

Tubulin is a heterodimeric protein consisting of alpha- and beta-monomers and plays a very important role in cell division. Tubulin dimers polymerize to form microtubules, which are required for cell divisions. Three dimensional structures of tubulin solved to date lack the final 15-20 amino acids of the C-termini of each subunit. These peptides are of interest because they are the primary locus of tubulin sequence heterogeneity and posttranslational modification of the protein. In polymerized tubulin, the C-terminal peptides are on the exterior of the microtubule and are implicated in the association of microtubules with other proteins in the cell. How these associations occur are unknown because little is known about the structure of the C-terminal peptides. The goal of this research is to develop tools that can be used to assess the carboxy termini of tubulin in vitro and in live cells.;We have utilized a very unique posttranslational modification of tubulin where the protein undergoes reversible tyrosination/detyrosination via the antagonistic action of tubulin carboxypeptidase (TCP) and tubulin tyrosine ligase (TTL). TTL is very specific for alpha-tubulin and tyrosine, however, it has been shown that TTL will accept tyrosine analogs that have modifications in the 3-position on the ring. One of these unnatural amino acids, 3-formyltyrosine, contains a reactive functional group that may be reacted with hydrazine-containing molecules to form a stable covalent bond.;Since 3-formyltyrosine and hydrazine based fluorophores are commercially unavailable, we have synthesized and characterized them in our laboratory. It was observed that the hydrazone formation between 3-formyltyrosine and the fluorophores resulted in a shift of the emission maximum to longer wavelength and increased the quantum yield as compared to starting materials. We found that the fluorophores react well at the physiological conditions making them suitable for our purpose.;The posttranslational incorporation of the 3-formyltyrosine into alpha-tubulin by TTL in cell free conditions was confirmed by immunoblotting and mass spectrometry. Chemical ligation of the fluorophore with tubulin containing 3-formyltyrosine was performed. The success and specificity of the fluorescent labeling were demonstrated by SDS-PAGE of the fluorescently-labeled protein, which showed a single fluorescent band that corresponds to alpha-tubulin. The bioconjugation reaction can be performed under physiological conditions that preserve the native structure of the protein (pH 6.9, temperature < 37°C, time < 2 hr). The tyrosine derivative and the synthetic fluorophores were found to be non-cytotoxic to PC-3 cells at low micromolar concentrations. Preliminary experiments showed that this reaction can be used to label microtubules under cellular environment when examined under confocal microscopy.;We conclude that this work demonstrates a successful utilization of bioorthogonal conjugation reaction to site-specifically label tubulin.