RESUMO
Over 20 years ago, the pyrrolysine encoding translation system was discovered in specific archaea. Our Review provides an overview of how the once obscure pyrrolysyl-tRNA synthetase (PylRS) tRNA pair, originally responsible for accurately translating enzymes crucial in methanogenic metabolic pathways, laid the foundation for the burgeoning field of genetic code expansion. Our primary focus is the discussion of how to successfully engineer the PylRS to recognize new substrates and exhibit higher in vivo activity. We have compiled a comprehensive list of ncAAs incorporable with the PylRS system. Additionally, we also summarize recent successful applications of the PylRS system in creating innovative therapeutic solutions, such as new antibody-drug conjugates, advancements in vaccine modalities, and the potential production of new antimicrobials.
Assuntos
Aminoacil-tRNA Sintetases , Código Genético , Lisina , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/genética , Lisina/metabolismo , Lisina/análogos & derivados , Lisina/química , Metano/análogos & derivados , Metano/metabolismo , Metano/química , Evolução Molecular , HumanosRESUMO
We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/química , Cromatografia de Fase Reversa/métodosRESUMO
The Orange Carotenoid Protein (OCP) regulates cyanobacterial photosynthetic activity through photoactivation in intense light. A hydrogen bonding network involving the keto-carotenoid oxygen and Y201 and W288 residues prevents the spontaneous activation of dark-adapted OCP. To investigate the role of the hydrogen bonds in OCP photocycling, we introduced non-canonical amino acids near the keto-carotenoid, particularly iodine at the meta-position of Y201. This modification significantly increased the yield of red OCP photoproducts, albeit with a shorter lifetime. Changes in tryptophan fluorescence during photocycling influenced by the presence of iodine near W288 revealed interactions between Y201 and W288 in the absence of the carotenoid in the C-domain. We propose that upon the relaxation of red states, a ternary complex with the carotenoid is formed. Analysis of spectral signatures and interaction energies indicates that the specific iodo-tyrosine configuration enhances interactions between the carotenoid and W288.
Assuntos
Iodo , Triptofano , Aminoácidos , Ligação de Hidrogênio , Proteínas de Bactérias/metabolismo , Fluorescência , Luz , Carotenoides/metabolismoRESUMO
All known organisms encode 20 canonical amino acids by base triplets in the genetic code. The cellular translational machinery produces proteins consisting mainly of these amino acids. Several hundred natural amino acids serve important functions in metabolism, as scaffold molecules, and in signal transduction. New side chains are generated mainly by post-translational modifications, while others have altered backbones, such as the ß- or γ-amino acids, or they undergo stereochemical inversion, e.g., in the case of D-amino acids. In addition, the number of non-canonical amino acids has further increased by chemical syntheses. Since many of these non-canonical amino acids confer resistance to proteolytic degradation, they are potential protease inhibitors and tools for specificity profiling studies in substrate optimization and enzyme inhibition. Other applications include in vitro and in vivo studies of enzyme kinetics, molecular interactions and bioimaging, to name a few. Amino acids with bio-orthogonal labels are particularly attractive, enabling various cross-link and click reactions for structure-functional studies. Here, we cover the latest developments in protease research with non-canonical amino acids, which opens up a great potential, e.g., for novel prodrugs activated by proteases or for other pharmaceutical compounds, some of which have already reached the clinical trial stage.
Assuntos
Antifibrinolíticos , Peptídeo Hidrolases , Aminoácidos , Endopeptidases , ProteóliseRESUMO
Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).
Assuntos
Poli-Hidroxialcanoatos , Pseudomonas chlororaphis , Pseudomonas/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Lipase/metabolismo , Poliésteres/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas chlororaphis/genética , Especificidade por SubstratoRESUMO
Orange Carotenoid Protein (OCP) is a water-soluble photoreceptor involved in photoprotection of cyanobacteria. The photoactive OCP contains a bound ketocarotenoid cofactor held in a protein matrix with a hydrogen bonding network. We have developed a system to replace essential residues of the photoactive OCP with non-canonical aromatic analogues that produce well-defined chemical or steric changes. Preliminary spectroscopic evaluation of the generated OCP variants demonstrates the potential of this "molecular surgery" to disentangle protein-chromophore interaction networks that are critical for photoreceptor function. In this way, the number and strength of key contacts with non-canonical amino acids could be controlled and manipulated. We have illustrated this principle here by replacing hydrogen bond donating residues with aromatic non-canonical amino acids that alter the state preference of OCP.
Assuntos
Aminoácidos Aromáticos , Cianobactérias , Aminoácidos/metabolismo , Aminoácidos Aromáticos/metabolismo , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Cianobactérias/metabolismoRESUMO
Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.
Assuntos
Proteínas de Bactérias/química , Fitocromo/química , Agrobacterium/química , Alanina/análogos & derivados , Alanina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Sítios de Ligação , Luz , Simulação de Dinâmica Molecular , Mutação , Nitrilas/química , Fitocromo/genética , Fitocromo/metabolismo , Fitocromo/efeitos da radiação , Conformação Proteica/efeitos da radiação , Eletricidade Estática , Estereoisomerismo , Tetrapirróis/química , Tetrapirróis/metabolismoRESUMO
Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on "iGEM-Synthetic Biology" offered at the Technische Universität Berlin as an example.
Assuntos
Pesquisa Biomédica , Biotecnologia/educação , Engenharia Genética , Organismos Geneticamente Modificados , Biologia Sintética/educação , Humanos , AprendizagemRESUMO
Circular dichroism is a conventional method for studying the secondary structures of peptides and proteins and their transitions. While certain circular dichroism features are characteristic of α-helices and ß-strands, the third most abundant secondary structure, the polyproline-II helix, does not exhibit a strictly conserved spectroscopic appearance. Due to its extended nature, the polyproline-II helix is highly accessible to the surrounding solvent; thus, the environment has a critical influence on the lineshape of the circular dichroism spectra of this structure. To showcase possible effects due to the medium, in this work, we report an experimental spectroscopic study of polyproline-II-forming oligomeric peptides in various environments: solvents, detergent micelles, and liposomes. Strikingly, the examination of an oligomeric peptide in a solvent series showed a remarkable 7 nm solvatochromic shift in the main negative band starting with hexafluoropropan-2-ol and moving to hexane. Furthermore, a previously predicted positive band below 200 nm was discovered in the spectra in nonpolar environments. In isotropic liposomes, the expected transition to the transmembrane state correlated with the appearance of a positive band at 228 nm. Our results demonstrate that changes in solvation should be taken into consideration when assessing the circular dichroism spectra of peptides expected to adopt the polyproline-II conformation. Although this precaution may complicate spectral analysis, characterization of solvent-induced spectral changes can generate new opportunities for testing the location of peptides in complex systems such as micelles or lipid bilayers.
Assuntos
Peptídeos/química , Alanina/química , Dicroísmo Circular , Conformação ProteicaRESUMO
Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology. The diversification of the repertoire of the genetic code has been achieved for amino acids with long and/or bulky side chains equipped with various bioorthogonal tags and useful spectral probes. Although ncAAs with relatively small side chains and similar properties are of great interest to biophysics, cell biology, and biomaterial science, they can rarely be incorporated into proteins. To address this gap, we report the engineering of PylRS variants capable of incorporating an entire library of aliphatic "small-tag" ncAAs. In particular, we performed mutational studies of a specific PylRS, designed to incorporate the shortest non-bulky ncAA (S-allyl-l-cysteine) possible to date and based on this knowledge incorporated aliphatic ncAA derivatives. In this way, we have not only increased the number of translationally active "small-tag" ncAAs, but also determined key residues responsible for maintaining orthogonality, while engineering the PylRS for these interesting substrates. Based on the known plasticity of PylRS toward different substrates, our approach further expands the reassignment capacities of this enzyme toward aliphatic amino acids with smaller side chains endowed with valuable functionalities.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Código Genético , Lisina/análogos & derivados , Methanosarcina barkeri/enzimologia , Engenharia de Proteínas , Aminoacil-tRNA Sintetases/genética , Proteínas Arqueais/metabolismo , Lisina/metabolismo , Especificidade por SubstratoRESUMO
A universal gain-of-function approach for the spatiotemporal control of protein activity is highly desirable when reconstituting biological modules inâ vitro. Here we used orthogonal translation with a photocaged amino acid to map and elucidate molecular mechanisms in the self-organization of the prokaryotic filamentous cell-division protein (FtsZ) that is highly relevant for the assembly of the division ring in bacteria. We masked a tyrosine residue of FtsZ by site-specific incorporation of a photocaged tyrosine analogue. While the mutant still shows self-assembly into filaments, dynamic self-organization into ring patterns can no longer be observed. UV-mediated uncaging revealed that tyrosine 222 is essential for the regulation of the protein's GTPase activity, self-organization, and treadmilling dynamics. Thus, the light-mediated assembly of functional protein modules appears to be a promising minimal-regulation strategy for building up molecular complexity towards a minimal cell.
Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Optogenética/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mathanococcus/metabolismo , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Nitrobenzenos/química , Tirosina/química , Raios UltravioletaRESUMO
Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4R)- and (4S)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.
RESUMO
Xenobiology is the science of estranged life forms. More specifically, this is an emergent technoscience that combines advances in genetic engineering with the design of biological systems based on unusual biochemistries delivered by chemical compounds of mostly anthropogenic origin. Xenobiology enables us to create and study strange new life forms, "aliens", not in the way science fiction books do it, but in terms of enlightened science, design, and engineering.
Assuntos
Biologia Sintética , Biotecnologia , Engenharia GenéticaRESUMO
In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re-engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans-sulfuration pathway. Our metabolic scheme operates inâ vivo, rescuing intermediates from core cell metabolism and combining them with small bio-orthogonal compounds. Our reprogrammed E. coli strain is capable of the in-cell production of l-azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology-based production.
Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica , Metionina/biossíntese , Metionina/análogos & derivados , Metionina/química , Estrutura MolecularRESUMO
In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as ß-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.
Assuntos
Aminoácidos Aromáticos , Aminoacil-tRNA Sintetases , Proteínas de Bactérias , Methanosarcina/enzimologia , Biossíntese de Proteínas , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Methanosarcina/genética , Mutagênese Sítio-Dirigida , Engenharia de ProteínasRESUMO
Catechols are a biologically relevant group of aromatic diols that have attracted much attention as mediators of adhesion of "bio-glue" proteins in mussels of the genus Mytilus. These organisms use catechols in the form of the noncanonical amino acid l-3,4-dihydroxyphenylalanine (DOPA) as a building block for adhesion proteins. The DOPA is generated post-translationally from tyrosine. Herein, we review the properties, natural occurrence, and reactivity of catechols in the design of bioinspired materials. We also provide a basic description of the mussel's attachment apparatus, the interplay between its different molecules that play a crucial role in adhesion, and the role of post-translational modifications (PTMs) of these proteins. Our focus is on the microbial production of mussel foot proteins with the aid of orthogonal translation systems (OTSs) and the use of genetic code engineering to solve some fundamental problems in the bioproduction of these bioadhesives and to expand their chemical space. The major limitation of bacterial expression systems is their intrinsic inability to introduce PTMs. OTSs have the potential to overcome these challenges by replacing canonical amino acids with noncanonical ones. In this way, PTM steps are circumvented while the genetically programmed precision of protein sequences is preserved. In addition, OTSs should enable spatiotemporal control over the complex adhesion process, because the catechol function can be masked by suitable chemical protection. Such caged residues can then be noninvasively unmasked by, for example, UV irradiation or thermal treatment. All of these features make OTSs based on genetic code engineering in reprogrammed microbial strains new and promising tools in bioinspired materials science.
Assuntos
Adesivos , Materiais Biomiméticos , Bivalves/química , Catecóis/química , Di-Hidroxifenilalanina/química , Engenharia Genética/métodos , Sequência de Aminoácidos , Animais , Ciência dos Materiais , Medicina , Processamento de Proteína Pós-TraducionalRESUMO
In contrast to many other water-soluble peptide arrangements, the formation of a triple helix in collagen proceeds inside out: polar glycyl residues form the interior, whereas nonpolar prolyl side chains constitute the exterior. In our work, we decided to exploit this aspect of the peptide architecture in order to create hyperstable collagen mimicking peptides (CMPs). The key element of this study is the environment. Given that the peptide assembles in a nonpolar medium, the collapse of the polar peptide backbone into the triple helix should become more favorable. Following this idea, we prepared CMPs based on hydrophobic proline analogues. The synthesis was performed by a combination of liquid- and solid-phase approaches: first, hexapeptides were prepared in solution, and then these were launched into conventional Fmoc-based peptide synthesis on a solid support. The resulting peptides showed an excellent signal of the triple helix in the model nonpolar solvent (octanol) according to circular dichroism observations. In a study of a series of oligomers, we found that the minimal length of the peptides required for triple helical assembly is substantially lower compared to water-soluble CMPs. Our results suggest further explorations of the CMPs in hydrophobic media; in particular, we highlight the suggestion that collagen could be converted into a membrane protein.
RESUMO
Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.
Assuntos
Bicamadas Lipídicas/metabolismo , Peptídeos/metabolismo , Flúor , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Peptídeos/síntese química , Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC-GCA-GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix. In the final phase (GCAU-phase), the amino acids were appended to the already existing architecture, enabling tertiary fold and membrane interactions. The whole scheme indicates strongly that the choice for the alanine core was done at the GCA-phase, while glycine and proline remained rudiments from the GC-phase. We suggest that the Protein World should rather be considered the Alanine World, as it predominantly relies on the alanine as the core chemical scaffold.
Assuntos
Alanina/genética , Aminoácidos/genética , Biossíntese de Proteínas , Proteínas/genética , Alanina/química , Aminoácidos/química , Animais , Evolução Molecular , Código Genético , Humanos , Dobramento de Proteína , Proteínas/químicaRESUMO
Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5-7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.