Extending the utility of enzymes for site-specific targeting of fluorescent probes

Abstract

Genetically encodable fluorescence reporters such as the green fluorescent protein (GFP) are useful for studying protein expression, localization, and dynamics in a variety of biological systems. GFP and its related variants, however, suffer from several drawbacks. Compared to chemical fluorophores, they are large, dim, and limited in other reporting capabilities. Super-bright chemical fluorophores such as the Alexa Fluor dyes and quantum dots, on the other hand, are not genetically encodable and so their cellular targeting is challenging. To address this challenge, the Ting Lab engineered E. coli lipoic acid ligase (LpIA) to site-specifically attach reporters onto a 13-amino acid ligase recognition peptide, conferring comparable targeting specificity to genetic encoding. This thesis is an extension of this work, to expand the repertoire of chemical fluorophores that can be targeted to cellular proteins by this technology. We describe the computational redesign of LpIA into a red fluorophore ligase, and the validation of this design by X-ray protein crystallography. We used this new technology for live-cell fluorescence imaging and super-resolution imaging. For the attachment of other fluorophores than cannot be directly bound by the enzyme we engineered LplA to incorporate functional handles that can be chemoselectively derivatized with fluorophores in a second step. In one example, LplA targeted a strained alkene to cellular proteins, which can subsequently react with dienophiles with exceptional kinetics. In another example, we show that LplA-targeted haloalkanes can efficiently recruit a modified haloalkane dehalogenase. These methods were used to label cells with diverse fluorophores, including quantum dots, and allowed tracking of single membrane proteins to study their lateral diffusion.