Reprogramming natural proteins using unnatural amino acids.

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

The studies discussed in this dissertation are aimed at the chemical-scale interactions involved in neuroreceptor structure and function. Unnatural amino acids were incorporated into several ligand-gated ion channels. Two different ionotropic glutamate receptors (iGluRs), the N-methyl-D-aspartate (NMDA) receptor and the α–amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptor were studied, along with an acetylcholine receptor - the nicotinic acetylcholine receptor (nAChR), and all were analyzed with electrophysiology–an assay of receptor function. In Chapter 2, a highly conserved tryptophan (Trp607) in the ion channel pore of the NMDA receptor was investigated for its role during extracellular Mg2+ block. Previous studies hypothesized that a cation-π interaction between NR2BW607 and Mg2+ contributed to the receptor blockade. However, our studies suggest that Trp607 is not involved in a cation-π interaction with Mg2+, instead it is a structural component of the pore. NR2B Trp607 acts as a steric “plug,” preventing Mg2+ permeation through the ion channel. These studies were the first to incorporate unnatural amino acids into a glutamate receptor, extending the scope of nonsense suppression methodology to a new class of neuroreceptors. Chapter 3 describes the incorporation of unnatural amino acids into the ligand binding domain (LBD) of NMDA and AMPA receptors. Previous structural studies of AMPA receptors established the overall topology of the LBD to be a clamshell, two domains clamp down around a central cleft. Further studies utilizing agonists that induce full receptor activation and partial receptor activation demonstrate a relationship between cleft closure and agonist efficacy, which is the ability to activate a receptor. Full agonists correlate with more cleft closure than partial agonists, which induce less cleft closure. To examine this relationship, we used unnatural amino acid mutagenesis to convert an NR2-conserved tyrosine to homotyrosine and an NR1 glutamine to homoglutamine, residues designed to disrupt clamshell closure by expanding the side chain without altering its functionality. The development of our functional probe demonstrates that the clamshell closure mechanism, previously shown for AMPA receptors, likely also applies to NMDA receptors, but to different degrees in the NR1 and NR2 subunits. Finally, in Chapter 4 we use unnatural amino acids, mutagenesis, and computational simulations to probe the binding interactions that are involved in agonist selectivity at the muscle-type nicotinic acetylcholine receptor. Acetylcholine (ACh) and nicotine, both agonists for nAChRs, have a high potency for neuronal receptors. However, nicotine is a weak agonist for the muscle-type nAChRs, yet the amino acids that contribute to the binding site remain the same between both types of receptors. These studies use mutagenesis and unnatural amino acids to introduce changes in the muscle-type receptor to increase nicotine potency. Although some of the mutations increase nicotine potency, none of the mutations result in a muscle-type receptor with nicotine potency as great as the neuronal receptors. A second set of studies generated a mouse muscle homology model and used molecular dynamics to simulate movements in the receptor with and without agonist bound. These structures demonstrate the importance of a hydrogen-bonding network that contributes to the pre-organization of the aromatic box.

Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.

Review: 112 refs.

The precise engineering of proteins in bacteria via the amber codon has been hampered by the poor incorporation of unnatural amino acid (UAA). Here we explored the amber assignment as a sense codon for UAA by CRISPRi targeting release factor 1 (RF1). Scanning of RF1 gene with sgRNAs identified target loci that differentiate RF1 repressions. Quantitation of RF1 repressions versus UAA incorporation indicated an increasing interrelation with the amber reassignment maximized upon RF1 knockdown to ~30%, disclosing the beneficial role of RF1 in amber assignment. However, further RF1 repression reversed this trend resulting from the detrimental effects on host cell growth, disclosing the harmful aspect of RF1 in reassignment of the amber codon. Our data indicate RF1 as a switch manipulating genetic code expansion and pave a direction via CRISPRi for precise engineering and efficient production of proteins in bacteria.

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Site-specific incorporation of unnatural amino acids (amino acid analogues) into proteins adds a new dimension to studies of protein structure and function. Here, we describe in detail the development of methods for site-specific incorporation of unnatural amino acids with novel chemical, physical and biological properties using specialized suppressor tRNAs alongside engineered aminoacyl-tRNA synthetases and ribosomes.

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

Various methods for characterizing binding forces as well as for monitoring and remote control of ion channels are still emerging. A recent innovation is the direct incorporation of unnatural amino acids (UAAs) with corresponding biophysical or biochemical properties, which are integrated using genetic code expansion technology. Minimal changes to natural amino acids, which are achieved by chemical synthesis of corresponding UAAs, are valuable tools to provide insight into the contributions of physicochemical properties of side chains in binding events. To gain unique control over the conformational changes or function of ion channels, a series of light-sensitive, chemically reactive and posttranslationally modified UAAs have been developed and utilized. Here, we present the existing UAA tools, their mode of action, their potential and limitations as well as their previous applications to Ca2+-permeable ion channels.

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.

New chemical and physical properties can be selectively introduced into proteins directly in live cells by genetically incorporating unnatural amino acids. The incorporation efficiency determines how effective such properties can be exploited and was very low in yeast. We developed a new method for efficient expression of orthogonal bacterial tRNA in yeast using polymerase III promoters that are cleaved from primary transcripts. In addition, a yeast strain deficient in nonsense-mediated mRNA decay was generated to prevent rapid degradation of target mRNA containing premature stop codons, which are the most frequently used to encode unnatural amino acids. These new strategies enabled a significant increase in yield of unnatural amino acid containing proteins from tens of micrograms to tens of milligrams per liter in yeast.

A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics.

Naturally, proteins generally consist of twenty natural amino acids (NAAs) as building blocks, which can form a nearly unlimited number of combinations by random combination to realize structural and functional diversity. Unfortunately, it is not enough using these natural blocks to generate proteins with specific characteristics. Therefore, incorporating unnatural amino acids (UNAAs) with some novel groups to expand the repertoire of structures and functions of proteins is becoming more and more popular [1]. Diverse bioreactivity from novel functional groups are unreachable to natural proteins, which opens gates for new protein engineering and provides new ways for fundamental research, therapeutics, and synthetic biology [2]. Incorporation of UNAA at a specific site has been used for studying protein structure and dynamics, characterizing protein-protein interactions, mimicking post-translational modifications of proteins like eukaryote, and synthesize novel products hard to be created by other methods, such as enzymes, biomaterials, and therapeutics [3].

The efficient, site-specific introductionof unnatural amino acidsinto proteins in mammalian cells is an outstanding challenge in realizingthe potential of genetic code expansion approaches. Addressing thischallenge will allow the synthesis of modified recombinant proteinsand augment emerging strategies that introduce new chemical functionalitiesinto proteins to control and image their function with high spatialand temporal precision in cells. The efficiency of unnatural aminoacid incorporation in response to the amber stop codon (UAG) in mammaliancells is commonly considered to be low. Here we demonstrate that tRNAlevels 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 mammaliancells. In addition, we engineer eRF1, that normally terminates translationon all three stop codons, to provide a substantial increase in unnaturalamino acid incorporation in response to the UAG codon without increasingreadthrough of other stop codons. By combining the optimized pyrrolysyl-tRNAsynthetase/tRNACUA expression system and an engineeredeRF1, we increase the yield of protein bearing unnatural amino acidsat a single site 17- to 20-fold. Using the optimized system, we produceproteins containing unnatural amino acids with comparable yields toa protein produced from a gene that does not contain a UAG stop codon.Moreover, the optimized system increases the yield of protein, incorporatingan unnatural amino acid at three sites, from unmeasurably low levelsup to 43% of a no amber stop control. Our approach may enable theefficient production of site-specifically modified therapeutic proteins,and the quantitative replacement of targeted cellular proteins withversions bearing unnatural amino acids that allow imaging or syntheticregulation of protein function.