Expressed Protein Ligation at Methionine: N‐Terminal Attachment of Homocysteine, Ligation, and Masking

Tanaka, Tomohiro; Wagner, Anne; Warner, John B.; Wang, Yanxin J.; Petersson, E. James

doi:10.1002/ange.201302065

Cited by 8 publications

(5 citation statements)

References 43 publications

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“…Logically, Cys alkylation has been combined with NCL to produce peptide bonds to pseudohomoglutamate, 639 pseudohomoglutamine, 405,640−643 pseudolysine, 644,645 or pseudoglycosylated residues (Figure 59, left). 646,647 Analogously, hCys thiol methylation yielded Xaa-Met junctions (Figure 59, right; entry 11, Table 17), 379,609,648,649 while hSec methylation enabled the production of selenomethionine peptides. 650 In the latter case, hSec methylation could be performed selectively in the presence of a Cys residue due to the large difference in pK a between thiols and selenols.…”

Section: Chemical Reviewsmentioning

confidence: 98%

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

et al. 2019

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The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro-and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O,S-, N,S-, or N,Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N,S-acyl migrations and selective dechalcogenation reactions.

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Section: Chemical Reviewsmentioning

confidence: 98%

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

et al. 2019

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“…334 Alternatively, Petersson and co-workers demonstrated that disulfide-protected homocysteine can be enzymatically installed on the N-terminus of proteins through the activity of a bacterial aminoacyl transferase, a method the authors used for a semisynthesis of α-synuclein. 335 Cysteine residues may be formally mutated to alanine through chemical desulfurization reactions (Figure 12c), which expands potential ligation junctions to this highly abundant amino acid. 336,337 As for the use of alkylation reactions postligation, such methods are incompatible with endogenous cysteine residues, which will also be chemically modified.…”

Section: Extensions and Alternatives To Native Chemical Ligationmentioning

confidence: 99%

Chemoenzymatic Semisynthesis of Proteins

2019

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Protein semisynthesisdefined herein as the assembly of a protein from a combination of synthetic and recombinant fragmentsis a burgeoning field of chemical biology that has impacted many areas in the life sciences. In this review, we provide a comprehensive survey of this area. We begin by discussing the various chemical and enzymatic methods now available for the manufacture of custom proteins containing noncoded elements. This section begins with a discussion of methods that are more chemical in origin and ends with those that employ biocatalysts. We also illustrate the commonalities that exist between these seemingly disparate methods and show how this is allowing for the development of integrated chemoenzymatic methods. This methodology discussion provides the technical foundation for the second part of the review where we cover the great many biological problems that have now been addressed using these tools. Finally, we end the piece with a short discussion on the frontiers of the field and the opportunities available for the future.

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“…Short-range interactions have typically been probed with tryptophan (Trp) or methoxycoumarin (Mcm) as the donor and Acd as the acceptor. Mcm can be incorporated on the amino acid sidechain through solid phase peptide synthesis or through ligation of a peptide fragment to a larger protein (Tanaka et al, 2013). However, it is generally more practical to incorporate Mcm by reaction with a sidechain, as we have previously reported for a maleimide derivative that can be reacted with Cys to give C Mcm (Ferrie et al, 2017;Jones et al, 2020).…”

Section: Acd Applicationsmentioning

confidence: 99%

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance‐dependent energy transfer

et al. 2023

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Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

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Expressed Protein Ligation at Methionine: N‐Terminal Attachment of Homocysteine, Ligation, and Masking

Cited by 8 publications

References 43 publications

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Chemoenzymatic Semisynthesis of Proteins

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance‐dependent energy transfer

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