Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Abstract

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

“…This was done by substituting other residues for Trp-37, which is located at the end of the lipoic acid-binding pocket and which acts as a "gatekeeper" residue. The mutations enlarge the active site volume and allow binding of substrates of smaller size and different shapes relative to lipoic acid (41)(42)(43)(44). However, some molecules are not utilized by LplA, although they are smaller than lipoate (41).…”

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…Tryptophan-37 is located at the end of the lipoic acid binding pocket and acts as a gatekeeper residue. The mutations enlarge the volume of the active site and allow binding of substrates having different sizes and shapes than lipoic acid (48)(49)(50)(51)(52).…”

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

“…Ideally the fluorescent probe should be non-fluorescent when unligated to the protein of interest, and unreacted probe should be easily washed out of cells. To this aim, more recent enzyme labeling strategies been developed to ensure complete and site-selective labeling, such as biotin ligase, transglutaminase, which labels a reactive glutamine within a specific recognition sequence, or a fluorophore ligase derived from Escherichia coli lipoic acid ligase (LplA), which has been engineered to catalyze rapid and specific intracellular ligation of synthetic 7-hydroxycoumarin, Pacific Blue, or resorufin to a 13mer transposable acceptor peptide within the protein of interest [150,151,152,153,154,155]. …”