Chayasith Uttamapinant

A fluorophore ligase for site-specific protein labeling inside living cells

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling.

We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1

The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.

Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells.

We demonstrate the evolution of the PylRS/tRNACUA pair for genetically encoding photocaged cysteine. By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, we establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. We demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.

Site-specific protein labeling using PRIME and chelation-assisted click chemistry

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min–3 h.

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.

Synthesis of 7‐Aminocoumarin by Buchwald–Hartwig Cross Coupling for Specific Protein Labeling in Living Cells

To enable minimally invasive studies of proteins in their native context, it is desirable to tag proteins with small, bright reporter groups. Recently, our lab described PRIME (probe incorporation mediated by enzymes) technology for such tagging.[1–3] An engineered variant of Escherichia coli lipoic acid ligase (LplA) is used to covalently attach a fluorescent substrate, such as 7-hydroxycoumarin, onto a 13-residue peptide-recognition sequence (called LAP, for ligase acceptor peptide) that is genetically fused to a protein of interest (POI; Scheme 1A). The targeting specificity is derived from the extremely high natural sequence specificity of LplA.[4] PRIME was used to label and visualize various LAP-tagged cytoskeletal and adhesion proteins in living mammalian cells.

Ribosome Subunit Stapling for Orthogonal Translation in E. coli

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.