Targeting the N terminus for site-selective protein modification.
TL;DR: This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each, along with their uses in a number of diverse biological applications.
Abstract: The formation of well-defined protein bioconjugates is critical for many studies and technologies in chemical biology. Tried-and-true methods for accomplishing this typically involve the targeting of cysteine residues, but the rapid growth of contemporary bioconjugate applications has required an expanded repertoire of modification techniques. One very powerful set of strategies involves the modification of proteins at their N termini, as these positions are typically solvent exposed and provide chemically distinct sites for many protein targets. Several chemical techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. A growing number of N-terminus-specific enzymatic ligation strategies have provided additional possibilities. This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each. Examples of specific N-terminal protein conjugates are provided, along with their uses in a number of diverse biological applications.
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TL;DR: This guide will help triage candidate methods for peptide alteration and will serve as a starting point for those seeking to solve long-standing challenges.
Abstract: Advances in bioconjugation and native protein modification are appearing at a blistering pace, making it increasingly time consuming for practitioners to identify the best chemical method for modifying a specific amino acid residue in a complex setting. The purpose of this perspective is to provide an informative, graphically rich manual highlighting significant advances in the field over the past decade. This guide will help triage candidate methods for peptide alteration and will serve as a starting point for those seeking to solve long-standing challenges.
320 citations
01 Mar 2019
TL;DR: The goal of this Review is to provide a window through which to view the many opportunities created by novel protein modification techniques‚ and to act as an initial guide to help scientists find direction and form ideas in an ever-growing field.
Abstract: Proteins constitute the majority of nature’s worker biomolecules. Designed for specific functions, complex tertiary structures make proteins ideal candidates for analysing natural systems and creating novel biological tools. Owing to both their large size and the need for proper folding, de novo synthesis of proteins has been quite a challenge, leading scientists to focus on modifying protein templates already provided by nature. Recently developed methods for protein modification fall into two broad categories: those that can modify the natural protein template directly and those that require genetic manipulation of the amino acid sequence before modification. The goal of this Review is not only to provide a window through which to view the many opportunities created by novel protein modification techniques‚ but also to act as an initial guide to help scientists find direction and form ideas in an ever-growing field. In addition to highlighting methods reported in the past 5 years, we aim to provide a broader sense of the goals and outcomes of protein modification and bioconjugation in general. While the main body of this paper comprises reactions involving the direct modification of expressed proteins, some further functionalization strategies as well as biological applications are also acknowledged. The discussion concludes by speculating which trends and discoveries will most likely come next in the field. Over the past 5 years, many novel site-selective protein modification techniques have been reported. Key features of these various strategies as well as prominent examples are discussed in this Review.
259 citations
TL;DR: It has been shown that photoredox catalysis can be used to specifically target protein C-termini toward decarboxylative-alkylation with Michael acceptors, and provides a blueprint toward the development of photored ox catalysis as a generic platform to target other redox-active side chains for native conjugation.
Abstract: The advent of antibody-drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
218 citations
TL;DR: It is shown that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.
Abstract: Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophi...
180 citations
TL;DR: A variety of promising strategies have appeared recently that address this grand challenge in chemical biology and yield native protein-based well-defined bioconjugations, specific labeling of endogenous proteins in various biological crude milieus, and the establishment of chemical proteomics as a new research area in protein science.
Abstract: Chemical modification of proteins provides powerful tools to realize a broad range of exciting biological applications, including the development of new classes of biopharmaceuticals and functional studies of individual proteins in complex biological systems. Numerous strategies for linking desired chemical probes with target proteins have been developed in the last two decades, with most exploiting genetic protein engineering and/or bio-orthogonal chemistry that utilizes unnatural amino acids incorporated into proteins. Modification of native proteins in test tubes and biological contexts by site-specific and target-selective approaches remains challenging because appropriate organic chemistry to carry out such modifications is currently limited. Nonetheless, a variety of promising strategies have appeared recently that address this grand challenge in chemical biology. These new chemistries yield native protein-based well-defined bioconjugations, specific labeling of endogenous proteins in various biological crude milieus, and the establishment of chemical proteomics as a new research area in protein science. In this Perspective, we focus on recent remarkable progress in chemistry for native protein modification. We survey chemical characteristics of the methods and describe briefly these advanced applications to address unsolved biological issues. Current limitations and future directions of this research field are also discussed.
180 citations
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TL;DR: The technique of native chemical ligation is employable for chemically synthesizing full length proteins as discussed by the authors, which are chemically identical to proteins produced by cell free synthesis, and can be refolded and/or oxidized to form native disulfide-containing protein molecules.
Abstract: Proteins of moderate size having native peptide backbones are produced by a method of native chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. The technique of native chemical ligation is employable for chemically synthesizing full length proteins.
3,347 citations
TL;DR: A Cu-free variant of click chemistry that can label biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction is reported.
Abstract: Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
1,628 citations
906 citations
TL;DR: The combination of new sequences and structures enables better reconstruction of their evolutionary heritage and illuminates unrecognized similarities within this diverse group of enzymes.
Abstract: Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in the diversity of reactions that they catalyze. New structural data have paved the way for targeted mutagenesis and mechanistic studies and have provided a framework for interpretation of those results. Together, these complementary approaches yield new insight into function, particularly in understanding the origins of substrate and reaction type specificity. The combination of new sequences and structures enables better reconstruction of their evolutionary heritage and illuminates unrecognized similarities within this diverse group of enzymes. The important metabolic roles of many PLP-dependent enzymes drive efforts to design specific inhibitors, which are now guided by the availability of comprehensive structural and functional databases. Better understanding of the function of this important group of enzymes is crucial not only for inhibitor design, but also for the design of improved protein-based catalysts.
805 citations
TL;DR: This work is funded by the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship).
Abstract: O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.
781 citations