Pyrrolysyl-tRNA Synthetase Mutant Research Articles

Machine Learning Enables Prediction of Pyrrolysyl-tRNA Synthetase Substrate Specificity.

Knowledge about the substrate scope for a given enzyme is informative for elucidating biochemical pathways and also for expanding applications of the enzyme. However, no general methods are available to accurately predict the substrate specificity of an enzyme. Pyrrolysyl-tRNA synthetase (PylRS) is a powerful tool for incorporating various noncanonical amino acids (NCAAs) into proteins, which enabled us to probe, image, rationally engineer, and evolve protein structure and function. However, the incorporation of a new NCAA typically requires the selection of large libraries of PylRS with randomized mutations at active sites, and this process requires multiple rounds of selection for each new substrate. Therefore, a single aminoacyl-tRNA synthetase with broad substrate promiscuity is ideal to facilitate widespread applications of the genetic NCAA incorporation technique. Herein, machine learning models were developed to predict the substrate specificity of PylRS to accept novel NCAAs that could be incorporated into proteins by three PylRS mutants. The models were built from a training set of 285 unique enzyme-substrate pairs of three PylRS mutants including IFRS, BtaRS, and MFRS against 95 NCAAs. The best BaggingTree (BT) model was then used for virtually screening a NCAAs library containing 1474 phenylalanine, tyrosine, tryptophan, and alanine analogues, and 156 NCAAs were predicted to be accepted by at least one of the three PylRS mutants. Then, 27 NCAAs including 24 positive and 3 negative substrates were experimentally tested for their activities, and 20 of the 24 positive substrates showed weak or strong activity and were accepted by at least one PylRS mutant, among which 11 NCAAs were never reported to be incorporated into proteins before. Three negative substrates did not show any activity. Experimental results suggested that the BT model provides a three-class classification accuracy of 0.69 and a binary classification accuracy of 0.86. This study expanded the substrate scope of three PylRS variants and provided a framework for developing machine learning models to predict substrate specificity of other PylRS variants.

A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds

As one of the most valuable tools for genetic code expansion, pyrrolysyl-tRNA synthetase (PylRS) is structurally related to phenylalanyl-tRNA synthetase (PheRS). By introducing mutations that mimic ligand interactions in PheRS into PylRS, we designed a PylRS mutant. This mutant, designated as oClFRS, recognizes a number of o-substituted phenylalanines for their genetic incorporation at amber codon. Its efficiency in catalyzing genetic incorporation of o-chlorophenylalanine (o-ClF) is better than that for Nε-tert-butyloxycarbonyl-lysine catalyzed by PylRS. The crystal structure of oClFRS bound with o-ClF shows that o-ClF binds deeply into a hydrophobic but catalytically inactive pocket in the active site and involves two halogen bonds to achieve strong interactions. The shift of o-ClF to a catalytically active position in the oClFRS active site will be necessary for its activation. This is the first reported aminoacyl-tRNA synthetase that involves two halogen bonds for ligation recognition and might represent an alternative route to develop aminoacyl-tRNA synthetase mutants that are selective for noncanonical amino acids over native amino acids.

Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-l-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions.

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion.

Genetic code expansion (GCE) is a powerful technique for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells, which is achieved through evolved aminoacyl-tRNA synthetase mutants. Stability is important for promoting enzyme evolution, and we found that many of the evolved synthetase mutants have reduced thermostabilities. In this study, we characterized two novel pyrrolysyl-tRNA synthetases (PylRSs) derived from thermophilic archaea: Methanosarcina thermophila (Mt) and Methanosarcina flavescens (Mf). Further study demonstrated that the wild-type PylRSs and several mutants were orthogonal and active in both Escherichia coli and mammalian cells and could thus be used for GCE. Compared with the commonly used M.barkeri PylRS, the wild-type thermophilic PylRSs displayed reduced GCE efficiency; however, some of the mutants, as well as some chimeras, outperformed their mesophilic counterparts in mammalian cell culture at 37 °C. Their better performance could at least partially be attributed to the fact that these thermophilic synthetases exhibit a threshold of enhanced stability against destabilizing mutations to accommodate structurally diverse substrate analogues. These were indicated by the higher melting temperatures (by 3-6 °C) and the higher expression levels that were typically observed for the MtPylRS and MfPylRS mutants relative to the Mb equivalents. Using histone H3 as an example, we demonstrated that one of the thermophilic synthetase mutants promoted the incorporation of multiple acetyl-lysine residues in mammalian cells. The enzymes developed in this study add to the PylRS toolbox and provide potentially better scaffolds for PylRS engineering and evolution, which will be necessary to meet the increasing demands for expanded substrate repertoire with better efficiency and specificity in mammalian systems.

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7.

Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nε-(7-azidoheptanoyl)-l-lysine (AzHeK) by amber codon in Escherichia coli for recombinant expression of a number of AzHeK-containing histone H3 proteins. We assembled in vitro acyl-nucleosomes from these recombinant acyl-H3 histones. All these acyl-nucleosomes contained an azide functionality that allowed quick click labeling with a strained alkyne dye for in-gel fluorescence analysis. Using these acyl-nucleosomes as substrates and click labeling as a detection method, we systematically investigated chromatin deacylation activities of SIRT7, a class III NAD+-dependent histone deacylase with roles in aging and cancer biology. Besides confirming the previously reported histone H3K18 deacylation activity, our results revealed that SIRT7 has an astonishingly high activity to catalyze deacylation of H3K36 and is also catalytically active to deacylate H3K37. We further demonstrated that this H3K36 deacylation activity is nucleosome dependent and can be significantly enhanced when appending the acyl-nucleosome substrate with a short double-stranded DNA that mimics the bridging DNA between nucleosomes in native chromatin. By overexpressing SIRT7 in human cells, we verified that SIRT7 natively removes acetylation from histone H3K36. Moreover, SIRT7-deficient cells exhibited H3K36 hyperacetylation in whole cell extracts, at rDNA sequences in nucleoli, and at select SIRT7 target loci, demonstrating the physiologic importance of SIRT7 in determining endogenous H3K36 acetylation levels. H3K36 acetylation has been detected at active gene promoters, but little is understood about its regulation and functions. Our findings establish H3K36 as a physiologic substrate of SIRT7 and implicate this modification in potential SIRT7 pathways in heterochromatin silencing and genomic stability.

Proximity‐Triggered Covalent Stabilization of Low‐Affinity Protein Complexes In Vitro and In Vivo

The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.

Multiple site-specific installations of Nε-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis.

Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.

Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.

Genetic Incorporation of Seven ortho-Substituted Phenylalanine Derivatives

Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.

A Genetically Encoded Acrylamide Functionality

Nε-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNACUAPyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Nε-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Nε-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.

Genetic Incorporation of Twelve meta-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant

When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum.

Together with tRNA(CUA)(Pyl), a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large para substituents into superfolder green fluorescent protein at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pairs.