Incorporation of a Highly Reactive Oxalyl Thioester-Based Interacting Handle into Proteins

Grain, Benjamin; Desmet, Rémi; Snella, Benoît; Melnyk, Oleg; Agouridas, Vangelis

doi:10.1021/acs.orglett.3c01846

Cited by 3 publications

(2 citation statements)

References 29 publications

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“…There is widespread current interest in the cellular biosynthesis of proteins and polypeptides whose backbones differ from those in extant proteins 1 . Even single-atom substitutions, such as the introduction of an ester 2–8 or thioester 9–11 in place of an amide, can promote new chemistry 12 , facilitate mechanistic studies 2 , and generate materials with emergent properties 13–15 . Small molecule synthetic chemists well-appreciate the impact of single atom substitutions 16 .…”

Section: Mainmentioning

confidence: 99%

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Roe,

Schissel,

Dover

et al. 2023

Preprint

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Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an α-amino acid or α-hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non-α-amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse α-hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an α-hydroxy acid monomer into a protein translatedin vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an α-peptide backbone directly into a β-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone β-amino acid linkage has been introduced site-selectively into a protein in a cell.

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Section: Mainmentioning

confidence: 99%

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Roe,

Schissel,

Dover

et al. 2023

Preprint

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show abstract

“…Total chemical synthesis, on the other hand, allows for full control of all amino acids. While conventional solid-phase peptide synthesis (SPPS) is limited in length, methods for dovetailing peptide fragments have been developed and enabled the preparation of full-length proteins. , The chemical synthesis of proteins has enabled the site-selective incorporation of defined post-translational modifications and consequently the study of protein variants hardly attainable by recombinant expression. − However, peptide ligation approaches are often sequence-dependent and can require cumbersome multistep syntheses, making the preparation of complex protein variants challenging. , In contrast, recent advances in rapid automated flow peptide synthesis (AFPS) allow the rapid chemical synthesis of whole proteins with desired modifications of up to 164 amino acids , or even whole protein complexes . This opens opportunities to engineer designer proteins with unprecedented precision, including proteins with defined conformations.…”

Section: Introductionmentioning

confidence: 99%

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets

Charalampidou,

Nehls,

Meyners

et al. 2024

ACS Cent. Sci.

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Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket�as confirmed by crystal structures� which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

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Protocol for protein modification using oxalyl thioester-mediated chemoselective ligation

Terzani,

Wang,

Rostami

et al. 2024

STAR Protocols

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Incorporation of a Highly Reactive Oxalyl Thioester-Based Interacting Handle into Proteins

Cited by 3 publications

References 29 publications

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets

Protocol for protein modification using oxalyl thioester-mediated chemoselective ligation

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Incorporation of a Highly Reactive Oxalyl Thioester-Based Interacting Handle into Proteins

Cited by 3 publications

References 29 publications

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets

Protocol for protein modification using oxalyl thioester-mediated chemoselective ligation

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Product

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Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages