Noncanonical Amino Acid Mutagenesis Research Articles

Proline Residues in the Transmembrane/Extracellular Domain Interface Loops Have Different Behaviors in 5-HT3 and nACh Receptors.

Cys-loop receptors are important drug targets that are involved in signaling in the nervous system. The binding of neurotransmitters in the extracellular region of these receptors triggers an allosteric activation mechanism, the full details of which remain elusive, although structurally flexible loops in the interface between the extracellular region of Cys-loop receptors and the pore-forming transmembrane domain are known to play an important role. Here we explore the roles of three largely conserved Pro residues in two of these loops, the Cys-loop and M2-M3 loop, in 5-HT3A and α7 nACh receptors. The data from natural and noncanonical amino acid mutagenesis suggest that in both proteins a Pro is essential in the Cys-loop, probably because of its enhanced ability to form a cis peptide bond, although other factors are also involved. The important characteristics of Pros in the M2-M3 loop, however, differ in these two receptors: in the 5-HT3 receptor, the Pros can be replaced by some charged amino acids resulting in EC50s similar to those of wild-type receptors, while such substitutions in the nACh receptor ablate function. Ala substitution at one of these Pros also has different effects in the two receptors. Thus, our data show that even highly conserved residues can have distinct behaviors in related Cys-loop receptors.

The Recent Progresses in Chemical Synthesis of Proteins with Site-Specific Lysine Post-translational Modifications

In the past two decades, a plethora of lysine (Lys) posttranslational modifications (PTMs) has been discovered on proteins, major groups are acylation, alkylation, and ubiquitination. Although considered biologically important, functional annotation of proteins with Lys PTMs has largely fallen behind the discovery. One grand challenge of characterizing proteins with PTMs is the procurement of homogenously modified proteins. To resolve this obstacle, sophisticated methods have been developed. These include total synthesis, semisynthesis that is based on native chemical ligation, expressed protein ligation, and enzyme-catalyzed peptide ligation, and the amber-suppression based noncanonical amino acid mutagenesis technique that may need to couple with follow-up bioorthogonal chemistry. This review summarizes currently identified significant PTMs and chemical biology methods for their installation in proteins. We hope that the current review will provide helpful insights and critical perspectives to this important research frontier.

Binding Interactions of NS6740, a Silent Agonist of the α7 Nicotinic Acetylcholine Receptor

The α7 nicotinic acetylcholine receptor (nAChR) is a potential drug target for the treatment of a number of neurologic and inflammatory disorders. Silent agonists are an emerging class of drugs that bind to the receptor but do not open the channel. Instead they shift the receptor to a desensitized state. Silent agonists may be able to target a subset of α7 nAChR-mediated signaling processes. Here we use noncanonical amino acid mutagenesis to characterize the binding to α7 by the silent agonist 1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)phenyl)furan-2-yl)methanone (NS6740). We find that, like α7 agonists, NS6740 forms a cation-π interaction with Y115 (TyrA). We also showed that NS6740 makes a novel hydrogen bond to TyrA. This interaction is necessary for the silent agonist activity of NS6740; when the hydrogen bond is blocked, silent agonist NS6740 converts to a conventional partial agonist and appreciably opens the channel in the absence of a positive allosteric modulator (EC50 150 nM). SIGNIFICANCE STATEMENT: Noncanonical amino acids were used to show that a hydrogen bond to tyrosine (Y115) is required for silent agonist activity of NS6740 at the α7 nicotinic acetylcholine receptor.

Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons.

Genetic incorporation of noncanonical amino acids has emerged as a powerful tool for the study of protein structure and function. While the three triplet nonsense codons have been widely explored, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acid mutagenesis. Here we demonstrated for the first time that two orthogonal quadruplet codons could be used to simultaneously encode two different noncanonical amino acids within a single protein in bacterial cells. To achieve this, we fine-tuned the interaction between aminoacyl-tRNA synthetase and tRNA, which afforded up to 21-fold improvement in quadruplet codon decoding efficiency. This work represents a significant step toward the use of multiple quadruplet codons for noncanonical amino acid mutagenesis. Simultaneous incorporation of two or more noncanonical amino acids is of significant importance for biological applications that can benefit from multiple unique functional groups, such as fluorescence resonance energy transfer and nuclear magnetic resonance studies, and ultimately for the synthesis of completely unnatural biopolymers as new biomaterials.

Generation of Stable Amber Suppression Cell Lines.

Noncanonical amino acid mutagenesis via amber suppression provides the means to tailor proteins inside living cells. A wide range of noncanonical amino acids have been incorporated using the Methanococcus pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/PylT) in mammalian cell systems in proof of principle experiments, for (1)minimal genetically encoded fluorescence or affinity tagging, (2) photo-control of enzymes, (3) genetically encoded posttranslational protein modifications. We have developed a general and efficient method to genomically integrate the PylRS/PylT amber suppression machinery using PiggyBac-mediated transposition. A general protocol for the generation of stable amber suppression cell lines is described here. Using the modular plasmid system, homogenous and highly efficient amber suppression in a wide range of cell lines can be achieved.

Generation of Intramolecular FRET Probes via Noncanonical Amino Acid Mutagenesis.

Förster resonance energy transfer (FRET) probes are powerful tools to monitor protein-protein interactions and enzyme activities in a spatiotemporal manner in live cells. Using a combination of noncanonical amino acid (ncAA) mutagenesis and bioorthogonal labeling, we have developed intramolecular FRET probes consisting of a fluorescent protein and an organic dye within an individual protein. Herein we present a general approach to establish intramolecular FRET probes for imaging of protein activity in live cells.

Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids.

By evolving the N-terminal domain of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNAPyl , a mutant clone displaying improved amber-suppression efficiency for the genetic incorporation of Nϵ -(tert-butoxycarbonyl)-l-lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ -acetyl-l-lysine and Nϵ -(4-azidobenzoxycarbonyl)-l-δ,ϵ-dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N-terminal domain of PylRS that was separated from its catalytic domain for charging tRNAPyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system-based noncanonical amino-acid mutagenesis.

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation.

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technology in mammalian cells, and highlights aspects of this nascent technology that need to be addressed to realize its full potential.

Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells.

We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents.

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

Insights into Gating Motions of GLIC via Perturbation of Critical Prolines with Non-Canonical Amino Acid Probes

Pentameric ligand-gated ion channels (pLGICs) are membrane proteins involved in fast synaptic transmission, and have been implicated in a number of neurodegenerative and psychological diseases in humans. These proteins have been the focus of extensive biochemical investigation, but high-resolution structures of mammalian receptors have only recently become available. The bacterial pLGIC Gloeobacter violaceus ligand-gated ion channel (GLIC), whose structure was first published in 2008, has served as a valuable model. There are now over 40 published structures obtained under various conditions, and these suggest specific conformational transitions that may occur during channel activation. If these gating motions are conserved throughout the pLGIC family, a detailed understanding of the process holds promise for the development of strategies to tune receptor activity. Here we discuss recent work in which we explore the role of prolines in the gating transitions of GLIC. Receptors were expressed in Xenopus oocytes, and examined using whole-cell electrophysiology. We first probed the importance of each proline residue in the receptor with an alanine scan. Pro119 in the Cys-loop, Pro198 and Pro203 in the M1 helix, and Pro299 in the M4 helix were sensitive to substitution. At these positions, we then used in vivo non-canonical amino acid mutagenesis with a range of proline analogs to determine specific structural requirements. The data revealed unique requirements at each position, and show that Pro119, Pro203 and Pro299 obey previously observed phenotypes for cis-preferring, bulge-inducing, and kink-inducing prolines respectively. However, an unusual phenotype was displayed by Pro198, a non-conserved site, and possible interpretations will be discussed.

Probing Binding Interactions of Agonists with the α6β2 Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is a ligand gated ion channel and a member of the Cys-loop family, which also contains receptors for serotonin, glycine, and GABA. There are twelve known neuronal subunits which pentamerize in different combinations to form various subtypes, each with a unique function, pharmacology and distribution in the brain. The α6β2-containing subtypes are found mainly in dopaminergic neurons and are thus important targets in the study of Parkinson's disease and addiction. Advanced knowledge of the binding site, which sits at the interface of α6 and β2, could lead toward design of agonists that specifically target this subtype. The α6L9'sβ2_(LFM/AAQA)L9's (α6β2‡) construct enabled heterologous expression and activation of a pure and stoichiometrically controlled population of α6β2 receptors in X. laevis oocytes. Currents were high enough in the α6β2‡ system to tolerate nonsense suppression-based non-canonical amino acid mutagenesis, which allowed for structure function studies between the receptor and several agonists. Initial structure-function studies probed for a cation-π interaction between agonists and the indole side chain of α6 TrpB. This is accomplished using an analog-tryptophan series whereby the negative electrostatic potential on the surface the indole ring was incrementally decreased via electron-withdrawing substituents such as fluorine and measuring the effect that has on binding. Results show that ACh makes a cation-π interaction, but surprisingly nicotine and TC-299423 do not. However, the latter two agonists do make a hydrogen bond interaction with the carbonyl of TrpB. This conclusion was made by substituting the adjacent amino acid with an alpha-hydroxy acid and measuring the corresponding shift in binding due to that amide-to-ester backbone mutation. The result was further quantified for nicotine in a double-mutant cycle analysis utilizing N’-methylnicotinium, which showed the interaction to have a coupling energy of ∼2 kcal/mol.

Performance of optimized noncanonical amino acid mutagenesis systems in the absence of release factor 1.

Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed in E. coli using UAG-suppression competes with termination mediated by release factor 1 (RF1). Recently, unconditional deletion of RF1 was achieved in a genomically recoded E. coli (C321), devoid of all endogenous UAG stop codons. Here we evaluate the efficiency of ncAA incorporation in this strain using optimized suppression vectors. Even though the absence of RF1 does not benefit the suppression efficiency of a single UAG codon, multi-site incorporation of a series of chemically distinct ncAAs was significantly improved.

Heterologous expression and nonsense suppression provide insights into agonist behavior at α6β2 nicotinic acetylcholine receptors

The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-β2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6β2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure–function studies between Trp149 – a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes – and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR.

Manipulating unconventional CH-based hydrogen bonding in a methyltransferase via noncanonical amino acid mutagenesis.

Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation's effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.

Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline

Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2–3-fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala.