- Azidohomoalanine: A conformationally sensitive IR probe of protein folding protein structure and electrostatics
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Highly sensitive: The azido analogue of methionine, azidohomoalanine (see picture), is shown to be a sensitive IR probe of protein structure, folding, and electrostatics, as demonstrated for ribosomal protein NTL9. It can be readily incorporated in to proteins, and the azido frequency is significantly blue-shifted in the thermally unfolded state.
- Taskent-Sezgin, Humeyra,Chung, Juah,Banerjee, Partha S.,Nagarajan, Sureshbabu,Dyer, R. Brian,Carrico, Isaac,Raleigh, Daniel P.
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- Coupling bioorthogonal chemistries with artificial metabolism: Intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins
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In this paper, we present a novel, single experiment methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-Azidohomoalanine from O-Acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-Auxotroph E. coli strain. In our system, the host's methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-Acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-Azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.
- Ma, Ying,Biava, Hernan,Contestabile, Roberto,Budisa, Nediljko,Di Salvo, Martino Luigi
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- Optimal linker length for small molecule PROTACs that selectively target p38α and p38β for degradation
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We report the design of hetero-bifunctional small molecules that selectively target p38α and p38β for degradation. These proteolysis targeted chimeras (PROTACs) are based on an ATP competitive inhibitor of p38α and p38β, which is linked to thalidomide analogues to recruit the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a copper catalyzed “click” reaction. We show that optimization of the linker length and composition is crucial for the degradation-inducing activity of these PROTACs. We provide evidence that these chemical compounds can induce degradation of p38α and p38β but no other related kinases at nanomolar concentrations in several mammalian cell lines. Accordingly, the PROTACs inhibit stress and cytokine-induced p38α signaling. Our compounds contribute to understanding the development of PROTACs, and provide a useful tool to investigate functions of the p38 MAPK pathway and its involvement in diseases.
- Donoghue, Craig,Cubillos-Rojas, Monica,Gutierrez-Prat, Nuria,Sanchez-Zarzalejo, Carolina,Verdaguer, Xavier,Riera, Antoni,Nebreda, Angel R.
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supporting information
(2020/07/06)
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- A concise route to L-azidoamino acids: L-azidoalanine, L-azidohomo-alanine and L-azidonorvaline
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A simple and highly efficient synthetic route to three homologous azidoamino acids, starting from inexpensive, commercially available, protected natural amino acids is reported. The products can be used to introduce bioorthogonal handles into proteins. Ge
- Roth, Stefanie,Thomas, Neil R.
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scheme or table
p. 607 - 609
(2010/09/18)
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- Nα-Fmoc-protected ω-azido- and ω-alkynyl-L- amino acids as building blocks for the synthesis of "clickable" peptides
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The growing interest in the 1,4-disubstituted-1,2,3-triazolyl moiety as an amide bond surrogate and its formation through very mild, chemoselective, and bioorthogonal CuI-catalyzed Huisgen 1,3-dipolar [3+2] cycloaddition of an alkynyl to an azi
- Isaad, Alexandra Le Chevalier,Barbetti, Francesca,Rovero, Paolo,D'Ursi, Anna Maria,Chelli, Mario,Chorev, Michael,Papini, Anna Maria
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experimental part
p. 5308 - 5314
(2009/06/18)
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