Advances in the mechanism and understanding of site-selective noncanonical amino acid incorporation

There are many approaches to introduce non-native functionality into proteins either translationally or post-translationally. When a noncanonical amino acid (NAA) is incorporated translationally, the host organism's existing translational machinery is relied upon to insert the amino acid by the same well-established mechanisms used by the host to achieve high fidelity insertion of its canonical amino acids. Research into the in vivo incorporation of NAAs has typically concentrated on evolving or engineering aminoacyl tRNA synthetases (aaRSs); however, new studies have increasingly focused on other members of the translational apparatus, for example entire ribosomes, in attempts to increase the fidelity and efficiency of incorporation of ever more structurally diverse NAAs. As the biochemical methods of NAA systems increase in complexity, it is informative to ask whether the 'rules' for canonical translation (i.e. aaRSs, tRNA, ribosomes, elongation factors, amino acid uptake, and metabolism) hold for NAA systems, or whether new rules are warranted. Here, recent advances in introducing novel chemical functionality into proteins are highlighted.     

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA^Pyl to create tRNA^Pyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N^ϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA^Pyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA^Pyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA^Pyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA^Pyl-opt should be an excellent replacement of wild-type tRNA^Pyl for future ncAA incorporation by PylRS enzymes.     

Synthesis of proteins with defined posttranslational modifications using the genetic noncanonical amino acid incorporation approach

Posttranslational modifications modulate the activities of most eukaryotic proteins and play a critical role in all aspects of cellular life. Understanding functional roles of these modifications requires homogeneously modified proteins that are usually difficult to purify from their natural sources. An emerging powerful tool for synthesis of proteins with defined posttranslational modifications is to genetically encode modified amino acids in living cells and incorporate them directly into proteins during the protein translation process. Using this approach, homogenous proteins with tyrosine sulfation, tyrosine phosphorylation mimics, tyrosine nitration, lysine acetylation, lysine methylation, and ubiquitination have been synthesized in large quantities. In this review, we provide a brief introduction to protein posttranslational modifications and the genetic noncanonical amino acid (NAA) incorporation technique, then discuss successful applications of the genetic NAA incorporation approach to produce proteins with defined modifications, and end with challenges and ongoing methodology developments for synthesis of proteins with other modifications.     

Variable denaturation of ovalbumin by incorporation of amino acid analogs

A novel firefly luciferin- enhanced luminescent procedure for the quantitation of horseradish peroxidase labels has been directly incorporated into established enzyme immunoassays. The procedure is rapid and sensitive and uses readily available reagents. Light emission from the enhanced reaction is high and relatively constant and thus easily measured. The luminescence procedure has been successfully incorporated into immunometric assays for rubella antibody and human IgE and into a competitive immunoassay for digoxin.     

Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids

In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP.     

Enzymes with noncanonical amino acids

Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.     

Unnatural amino acid incorporation into virus-like particles

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qbeta capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qbeta suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.     

Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

Kinetics of amino acid incorporation into serum proteins

1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q₁₀ was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q₁₀ of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues.     

Characterisation of amino acid incorporation by subcellular fractions from sterile beet disks

Summary The optimum concentrations of leucine, ATP, GTP and Mg²⁺ ion for the incorporation of leucine into protein by the microsomal fraction isolated from sterile disks of red beetroot are 0.06 mM, 5 mM, 0.5 mM, and 12 mM respectively. Incorporated ¹⁴C-leucine does not exchange with an excess of soluble-¹²C-leucine. Incorporation into protein is partly dependent on the addition of a high speed supernatant fraction which incorporates leucine into a product with the properties of aminoacyl RNA. Addition of polyuridylic acid to microsomes isolated from fresh disks stimulates the incorporation of phenylalanine into protein nine-fold but has no effect on leucine incorporation. Polyuridylic acid — stimulated incorporation is not inhibited by chloramphenicol. Preincubation of fresh microsomes with trypsin does not increase their activity. These results suggest that the low activity of fresh microsomes may be due to a lack of messenger RNA. The mitochondrial fraction shows a rise and fall in leucine-incorporating ability during aging similar to that shown by the microsomal fraction. Studies with inhibitors suggest that about 25% of this incorporation is due to the mitochondria themselves, the rest being attributable to large microsomes. Fractions isolated from disks aged under non-sterile conditions show large incorporations of leucine which are not dependent on an added energy source. This result confirms the importance of using aseptic techniques when studying the aging of storage tissue disks.