Evolving the Substrate Specificity of O⁶-Alkylguanine-DNA Alkyltransferase through Loop Insertion for Applications in Molecular Imaging

Abstract

We introduce a strategy for evolving protein substrate specificity by the insertion of random amino acid loops into the protein backbone. Application of this strategy to human O6-alkylguanine-DNA alkyltransferase (AGT) led to the isolation of mutants that react with the non-natural substrate O6-propargylguanine. Libraries generated by conventional random or targeted saturation mutagenesis, by contrast, did not yield any mutants with activity towards this new substrate. The strategy of loop insertion to alter enzyme specificity should be general and applicable to other classes of proteins. An important application of the isolated AGT mutant is in molecular imaging, where the mutant and parental AGTs are used to label two different AGT fusion proteins with different fluorophores in the same living cell or in vitro . This allowed the establishment of fluorescence-based assays to detect protein-protein interactions and measure enzymatic activities.

Inducing and Sensing Protein–Protein Interactions in Living Cells by Selective Cross‐linking

“…However, when the goal of the experiment is the sensing of the proximity of two different proteins, the formation of homodimers can serve as an internal standard (see above). The recent development of an AGT mutant that reacts with a substrate that is not recognized by the AGT mutants used here [15] and the introduction of a self-labeling protein tag based on mutants of a bacterial dehalogenase [16] could furthermore lead to CoDis that allow a directional and covalent dimerization of fusion proteins.…”

A Neo-Substrate that Amplifies Catalytic Activity of Parkinson’s-Disease-Related Kinase PINK1

“…In one example, the deubiquitinase UCH-L5 can hydrolyze larger ubiquitin chains only when a >14 AA loop is present in the active site (Zhou et al, 2012). In another example, protein engineering of alkyl guanine DNA alkyltransferase through insertion of a loop into the active site allows for recognition of an enlarged O 6 -modified guanine substrate not accepted by the enzyme without the loop insertion (Heinis et al, 2006). In light of these findings, the three insertions in PINK1’s adenine binding N-terminal subdomain led us to believe that PINK1 might also exhibit altered substrate specificity.…”

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters

“…Faced with these limitations, chemical biologists designed genetically encoded protein tags that bind synthetic fluorophore ligands . These include tags based on DNA alkyl transferases (SNAP and CLIP tags), dehalogenases (HaloTag), dihydrofolate reductase (TMP tag), and antibody fragments (e.g., fluorogen‐activating proteins). The introduction of synthetic chemical reporters improves the palette of fluorophores and enables new applications in cellular imaging, such as multicolor super‐resolution imaging .…”