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1.
Chem Rev ; 124(10): 6592-6642, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38691379

RESUMO

Reversible phosphorylation is a fundamental mechanism for controlling protein function. Despite the critical roles phosphorylated proteins play in physiology and disease, our ability to study individual phospho-proteoforms has been hindered by a lack of versatile methods to efficiently generate homogeneous proteins with site-specific phosphoamino acids or with functional mimics that are resistant to phosphatases. Genetic code expansion (GCE) is emerging as a transformative approach to tackle this challenge, allowing direct incorporation of phosphoamino acids into proteins during translation in response to amber stop codons. This genetic programming of phospho-protein synthesis eliminates the reliance on kinase-based or chemical semisynthesis approaches, making it broadly applicable to diverse phospho-proteoforms. In this comprehensive review, we provide a brief introduction to GCE and trace the development of existing GCE technologies for installing phosphoserine, phosphothreonine, phosphotyrosine, and their mimics, discussing both their advantages as well as their limitations. While some of the technologies are still early in their development, others are already robust enough to greatly expand the range of biologically relevant questions that can be addressed. We highlight new discoveries enabled by these GCE approaches, provide practical considerations for the application of technologies by non-GCE experts, and also identify avenues ripe for further development.


Assuntos
Código Genético , Fosforilação , Fosfoaminoácidos/metabolismo , Fosfoaminoácidos/química , Fosfoaminoácidos/genética , Proteínas/metabolismo , Proteínas/química , Proteínas/genética , Humanos
2.
Bioconjug Chem ; 34(12): 2243-2254, 2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-38047550

RESUMO

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/química , Proteínas/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Código Genético , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo
3.
Bio Protoc ; 13(21): e4861, 2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-37969748

RESUMO

While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH2) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This "PermaPhos" expression system uses the E. coli BL21(DE3) ΔserC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein-protein interaction networks from cell lysates or transfected cells. Key features • Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway. • Protein expression uses standard Terrific Broth (TB) media and requires three days to complete. • C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein. • Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.

5.
bioRxiv ; 2023 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-37693391

RESUMO

Receptor tyrosine kinase signaling is characterized by complex webs of interconnected pathways that regulate diverse cellular functions. The complexity of signaling is a barrier to understanding the pathways that control any particular function. In this work, we use a novel combination of approaches and a new click chemistry probe to determine the role of one pathway in regulating cell surface expression of an ion channel and a receptor tyrosine kinase. We applied an optogenetic approach to uncouple activation of the PI3K pathway from other pathways downstream of RTK activation. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the PM in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

6.
J Mol Biol ; 435(17): 168193, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37406927

RESUMO

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.


Assuntos
Proteínas de Ligação ao Cálcio , Cálcio , Disferlina , Proteínas de Membrana , Fosfatidilinositol 4,5-Difosfato , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Disferlina/química , Disferlina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios C2 , Ligação Proteica , Fosfatidilinositol 4,5-Difosfato/química
7.
J Am Chem Soc ; 145(27): 14608-14620, 2023 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-37364003

RESUMO

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.


Assuntos
Aminoácidos , Compostos Heterocíclicos , Animais , Humanos , Aminoácidos/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Células HEK293 , Proteínas/química , Mamíferos/metabolismo
8.
ACS Cent Sci ; 9(4): 816-835, 2023 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-37122473

RESUMO

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH2, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

9.
Protein Sci ; 32(5): e4640, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37051694

RESUMO

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.


Assuntos
Aminoacil-tRNA Sintetases , Histidina , Histidina/genética , Lisina/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/química , Methanosarcina/genética , Methanosarcina/metabolismo
10.
Cell Chem Biol ; 30(4): 343-361, 2023 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-36977415

RESUMO

The ability to selectively modify proteins at two or more defined locations opens new avenues for manipulating, engineering, and studying living systems. As a chemical biology tool for the site-specific encoding of non-canonical amino acids into proteins in vivo, genetic code expansion (GCE) represents a powerful tool to achieve such modifications with minimal disruption to structure and function through a two-step "dual encoding and labeling" (DEAL) process. In this review, we summarize the state of the field of DEAL using GCE. In doing so, we describe the basic principles of GCE-based DEAL, catalog compatible encoding systems and reactions, explore demonstrated and potential applications, highlight emerging paradigms in DEAL methodologies, and propose novel solutions to current limitations.


Assuntos
Aminoácidos , Proteínas , Proteínas/metabolismo , Aminoácidos/química , Código Genético
11.
bioRxiv ; 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36747808

RESUMO

Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans -cyclooctene (sTCO)-functionalized nitroxides-including a gem -diethyl-substituted nitroxide with enhanced stability in cells-with rate constants that can exceed 10 6 M -1 s -1 . The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro . Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

12.
Nat Commun ; 14(1): 59, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36599844

RESUMO

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.


Assuntos
Aminoacil-tRNA Sintetases , Fenilalanina , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Halogenação , Fenilalanina/metabolismo , RNA de Transferência/metabolismo , Humanos , Células HEK293
13.
Protein Sci ; 32(3): e4574, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36691781

RESUMO

14-3-3 proteins are central hub regulators of hundreds of phosphorylated "client" proteins. They are subject to over 60 post-translational modifications (PTMs), yet little is known how these PTMs alter 14-3-3 function and its ability to regulate downstream signaling pathways. An often neglected, but well-documented 14-3-3 PTM found under physiological and immune-stimulatory conditions is the conversion of tyrosine to 3-nitro-tyrosine at several Tyr sites, two of which are located at sites considered important for 14-3-3 function: Y130 (ß-isoform numbering) is located in the primary phospho-client peptide-binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14-3-3/client complex formation and client regulation. By genetically encoding 3-nitro-tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14-3-3 structure, function, and client complexation. The 1.5 Å resolution crystal structure of 14-3-3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14-3-3. But, in contrast to other client-binding deficient variants, it did not localize to the nucleus. The 1.9 Å resolution structure of 14-3-3 nitrated at Y213 revealed unusual flexibility of its C-terminal α-helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest that nitration of 14-3-3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14-3-3 interactome.


Assuntos
Proteínas , Tirosina , Humanos , Tirosina/química , Proteínas/química , Nitratos/química , Nitratos/metabolismo
14.
Protein Sci ; 32(2): e4560, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36585836

RESUMO

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates-enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13 C- and 15 N-labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and there is mounting evidence that Ser-16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13 C-, 15 N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13 C-, 15 N-labeled media, and protein expression was induced with isopropyl ß-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional 31 P NMR spectra, comparison of 1 H-15 N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such 13 C-, 15 N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.


Assuntos
Amelogenina , Escherichia coli , Código Genético , Proteínas Recombinantes , Serina , Animais , Camundongos , Amelogenina/genética , Amelogenina/química , Amelogenina/metabolismo , Escherichia coli/metabolismo , Código Genético/genética , Código Genético/fisiologia , Fosfosserina , Proteínas Recombinantes/genética , Proteínas Recombinantes/química
15.
Bio Protoc ; 12(21)2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36505030

RESUMO

This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆ serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For "truncation-free" expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆ A ∆ fabR ∆ serB , though use of the standard RF1-containing BL21(DE3) ∆ serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100-200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein-protein interactions. Graphical abstract.

16.
ACS Chem Biol ; 17(12): 3458-3469, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36383641

RESUMO

Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) and Methanosarcina barkeri (Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 µM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm/Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNAPyl GCE platform.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Humanos , Células HEK293 , Lisina/química , Aminoacil-tRNA Sintetases/metabolismo , Methanosarcina/genética , Methanosarcina/metabolismo , RNA de Transferência/genética , Tirosina
17.
ACS Chem Biol ; 17(12): 3470-3477, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36395426

RESUMO

A recently developed genetic code expansion (GCE) platform based on the pyrrolysine amino-acyl tRNA synthetase (PylRS)/tRNAPyl pair from Methanomethylophilus alvus (Ma) has improved solubility and lower susceptibility to proteolysis compared with the homologous and commonly used Methanosarcina barkeri (Mb) and M. mazei (Mm) PylRS GCE platforms. We recently created two new Ma PylRS variants for the incorporation of the fluorescent amino acid, acridonyl-alanine (Acd), into proteins at amber codons: one based on "transplanting" active site mutations from an established high-efficiency Mb PylRS and one that was de novo selected from a library of mutants. Here, we present the crystal structures of these two Ma PylRS variants with Acd/ATP bound to understand why the "active site transplant" variant (Acd-AST) displayed 6-fold worse Acd incorporation efficiency than the de novo selected PylRS (called Acd-RS1). The structures reveal that the Acd-AST binding pocket is too small and binds the three-ring aromatic Acd in a distorted conformation, whereas the more spacious Acd-RS1 active site binds Acd in a relaxed, planar conformation stabilized by a network of solvent-mediated hydrogen bonds. The poor performance of the AST enzyme is ascribed to a shift in the Ma PylRS ß-sheet framework relative to that of the Mb enzyme. This illustrates a general reason why "active site transplantation" may not succeed in creating efficient Ma PylRSs for other noncanonical amino acids. This work also provides structural details that will help guide the development of future Ma PylRS/tRNAPyl GCE systems via de novo selection or directed evolution methods.


Assuntos
Aminoacil-tRNA Sintetases , Euryarchaeota , Especificidade por Substrato , Aminoacil-tRNA Sintetases/metabolismo , Lisina/química , RNA de Transferência/genética , Methanosarcina barkeri/genética , Aminoácidos , Methanosarcina/genética , Methanosarcina/metabolismo
18.
Nat Sci (Weinh) ; 2(4)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36440454

RESUMO

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer (FRET) is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ~9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn-on ratios to ~14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.

19.
J Biol Chem ; 298(12): 102613, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36265582

RESUMO

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.


Assuntos
COVID-19 , Humanos , Fosfosserina/metabolismo , SARS-CoV-2/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/química , Serina/genética , Serina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo
20.
Sci Adv ; 8(18): eabm6909, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35522749

RESUMO

Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups-site-specifically inserted by genetic code expansion at Nb surface sites-are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti-SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.

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