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1.
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
2.
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
3.
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
4.
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.

5.
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.

6.
ACS Chem Biol ; 16(11): 2612-2622, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34590824

RESUMO

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically defined protein-protein cross-linking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.


Assuntos
Aminoácidos/química , Proteínas/química , Alcenos/química , Alcinos/química , Aminas , Escherichia coli/genética , Corantes Fluorescentes/química , Ligação Proteica
7.
J Am Chem Soc ; 143(18): 7154-7163, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33914511

RESUMO

During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i.e., ligation efficiency). Here, the connection between ligation reaction efficiency and the retention of enzyme structure and activity was investigated by leveraging the extremely fast reaction of strained trans-cyclooctene (sTCOs) and tetrazines (Tet). Remarkably, upon immobilization via Tet-sTCO chemistry, carbonic anhydrase (CA) retained 77% of its solution-phase activity, while immobilization via less efficient reaction chemistries, such as thiol-maleimide and azide-dibenzocyclooctyne, led to activity retention of only 46% and 27%, respectively. Dynamic single-molecule fluorescence tracking methods further revealed that longer surface search distances prior to immobilization (>0.5 µm) dramatically increased the probability of CA unfolding. Notably, the CA distance to immobilization was significantly reduced through the use of Tet-sTCO chemistry, which correlated with the increased retention of structure and activity of immobilized CA compared to the use of slower ligation chemistries. These findings provide an unprecedented insight into the role of ligation reaction efficiency in mediating the exploration of denaturing hotspots on surfaces by enzymes, which, in turn, may have major ramifications in the creation of functional biohybrid materials.


Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Conformação Proteica , Desdobramento de Proteína , Propriedades de Superfície
8.
J Mol Biol ; 432(16): 4690-4704, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32569745

RESUMO

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/metabolismo , Fatores de Terminação de Peptídeos/deficiência , Tirosina/análogos & derivados , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Domínio Catalítico , Códon de Terminação , Simulação por Computador , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli , Engenharia Genética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Tirosina/metabolismo
9.
ACS Appl Mater Interfaces ; 11(40): 36391-36398, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525993

RESUMO

Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. We present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained trans-cyclooctene reaction with genetic code expansion to site-specifically place the tetrazine into a protein. For the first time, we use this strategy to immobilize defined amounts of oriented proteins onto beads and flat surfaces in under 5 min at submicromolar concentrations without compromising activity. This approach opens the door to generating and studying diverse protein-based biomaterials that are much more precisely defined and characterized, providing a greater ability to engineer properties across a wide range of applications.


Assuntos
Proteínas Imobilizadas/metabolismo , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Ciclo-Octanos/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Moleculares
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