Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

The lanthanides (Ln3+), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln3+ ion. Additionally, current Ln3+ chelator syntheses complicate efforts to directly incorporate Ln3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p-azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu2+-free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes.

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Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

PLoS ONE ◽

10.1371/journal.pone.0209726 ◽

2019 ◽

Vol 14 (3) ◽

pp. e0209726 ◽

Cited By ~ 2

Author(s):

Stephanie C. Bishop ◽

Robert Winefield ◽

Asokan Anbanandam ◽

Jed N. Lampe

Keyword(s):

Click Chemistry ◽

Small Molecule ◽

Aqueous Synthesis

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Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

10.1242/prelights.9300 ◽

2019 ◽

Author(s):

Zhang-He Goh

Keyword(s):

Click Chemistry ◽

Small Molecule ◽

Aqueous Synthesis

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High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Applied and Environmental Microbiology ◽

10.1128/aem.03417-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1718-1725 ◽

Cited By ~ 16

Author(s):

Masaomi Minaba ◽

Yusuke Kato

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Unnatural Amino Acids ◽

Mammalian Cells ◽

Expression System ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Translational Efficiency ◽

Site Specific ◽

Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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Biomolecular simulation based machine learning models accurately predict sites of tolerability to the unnatural amino acid acridonylalanine

Scientific Reports ◽

10.1038/s41598-021-97965-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sam Giannakoulias ◽

Sumant R. Shringari ◽

John J. Ferrie ◽

E. James Petersson

Keyword(s):

Machine Learning ◽

Amino Acid ◽

Unnatural Amino Acids ◽

Hydrophobic Interactions ◽

Score Function ◽

Unnatural Amino Acid ◽

Protein Yield ◽

Site Specific ◽

Score Functions ◽

Biological Phenomena

AbstractThe incorporation of unnatural amino acids (Uaas) has provided an avenue for novel chemistries to be explored in biological systems. However, the successful application of Uaas is often hampered by site-specific impacts on protein yield and solubility. Although previous efforts to identify features which accurately capture these site-specific effects have been unsuccessful, we have developed a set of novel Rosetta Custom Score Functions and alternative Empirical Score Functions that accurately predict the effects of acridon-2-yl-alanine (Acd) incorporation on protein yield and solubility. Acd-containing mutants were simulated in PyRosetta, and machine learning (ML) was performed using either the decomposed values of the Rosetta energy function, or changes in residue contacts and bioinformatics. Using these feature sets, which represent Rosetta score function specific and bioinformatics-derived terms, ML models were trained to predict highly abstract experimental parameters such as mutant protein yield and solubility and displayed robust performance on well-balanced holdouts. Model feature importance analyses demonstrated that terms corresponding to hydrophobic interactions, desolvation, and amino acid angle preferences played a pivotal role in predicting tolerance of mutation to Acd. Overall, this work provides evidence that the application of ML to features extracted from simulated structural models allow for the accurate prediction of diverse and abstract biological phenomena, beyond the predictivity of traditional modeling and simulation approaches.

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Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.727406 ◽

2021 ◽

Vol 13 ◽

Author(s):

Alexander Kuhlemann ◽

Gerti Beliu ◽

Dieter Janzen ◽

Enrica Maria Petrini ◽

Danush Taban ◽

...

Keyword(s):

Genetic Code ◽

Click Chemistry ◽

Unnatural Amino Acids ◽

Super Resolution ◽

Receptor Expression ◽

Fluorescence Labeling ◽

Site Specific ◽

Gaba A ◽

Genetic Code Expansion ◽

Super Resolution Microscopy

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

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Faculty Opinions recommendation of Site-specific protein immobilization using unnatural amino acids.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.723878764.793522625 ◽

2016 ◽

Author(s):

Morten Meldal ◽

Sanne Schoffelen

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Protein Immobilization ◽

Specific Protein ◽

Site Specific

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A Simple and Efficient Method to Generate Dual Site-Specific Conjugation ADCs with Cysteine Residue and an Unnatural Amino Acid

Bioconjugate Chemistry ◽

10.1021/acs.bioconjchem.1c00134 ◽

2021 ◽

Author(s):

Lin Zhang ◽

Zewei Wang ◽

Zhiyuan Wang ◽

Fang Luo ◽

Mingfeng Guan ◽

...

Keyword(s):

Amino Acid ◽

Efficient Method ◽

Cysteine Residue ◽

Unnatural Amino Acid ◽

Site Specific ◽

Dual Site

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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