ACS Publications. Most Trusted. Most Cited. Most Read
Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Am. Chem. Soc. 2024, 146, 49, 33309-33315
  • Open Access
Communication

Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme
Click to copy article linkArticle link copied!

  • Jo-Chu Tsou
    Jo-Chu Tsou
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Jo-Chu Tsou
  • Chun-Ju Tsou
    Chun-Ju Tsou
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
    More by Chun-Ju Tsou
  • Chun-Hsiung Wang
    Chun-Hsiung Wang
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Chun-Hsiung Wang
  • An-Li A. Ko
    An-Li A. Ko
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by An-Li A. Ko
  • Yi-Hui Wang
    Yi-Hui Wang
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Yi-Hui Wang
    Orcidhttps://orcid.org/0009-0002-2616-4129
  • Huan-Hsuan Liang
    Huan-Hsuan Liang
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
    More by Huan-Hsuan Liang
  • Jia-Cheng Sun
    Jia-Cheng Sun
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Jia-Cheng Sun
  • Kai-Fa Huang
    Kai-Fa Huang
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Kai-Fa Huang
  • Tzu-Ping Ko
    Tzu-Ping Ko
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Tzu-Ping Ko
    Orcidhttps://orcid.org/0000-0003-1794-2638
  • Shu-Yu Lin
    Shu-Yu Lin
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    More by Shu-Yu Lin
  • Yane-Shih Wang*
    Yane-Shih Wang
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
    *Email: [email protected], [email protected]
    More by Yane-Shih Wang
    Orcidhttps://orcid.org/0000-0003-2356-9577
Open PDFSupporting Information (1)

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2024, 146, 49, 33309–33315
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.4c14446
Published November 5, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine’s moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole’s electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin’s B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

This publication is licensed under

CC-BY 4.0 .
  • cc licence
  • by licence
Copyright © 2024 The Authors. Published by American Chemical Society

Subjects

what are subjects

Post-translational protein modifications (PTMs) are highly regulated and efficient mechanisms involved in coordinating most if not all known cellular processes. (1) Being able to biochemically reproduce PTMs both chemo- and site-selectively, (2) as nature would, would serve a broad range of applications, specifically in probing proteins and producing homogeneous bioconjugates (3) such as antibody– and protein–drug conjugates for therapeutic use. (4) To achieve this in proteins, biocatalysis platforms supported by the development of novel, efficient enzymes are much in need. (5) While it is common to target the highly nucleophilic lysine and cysteine side chains to elicit chemoselectivity, their higher reactivity entails lower selectivity for residues to be targeted in a site-selective manner. (2) Moreover, modifying residues that often host a vast array of native PTMs is likely to compromise protein activity. (1)

Targeting of the less yet still nucleophilic and also less frequently modified histidine imidazole side chain (6,7) (that may still be, for instance, phosphorylated for mediating protein–protein interactions (8) or methylated for histone epigenetic regulation (9)) (Scheme 1) can therefore be of great interest for the purposes of generating biologically active, homogeneous products, with modifications more likely to be bioorthogonal to native PTMs. Histidine’s similar abundance (∼2%) to cysteine in proteins (10) also poses it as a desirable target for modification when presented on protein surfaces for temporary functional regulation, considering its various indispensable roles in catalyzing nucleophilic reactions, (11) coordinating metals, (6) hydrogen bonding, and transferring protons within enzymes. (12) In this study, we thus aim to design a site-selective histidine modification biocatalysis platform inspired by natural PTM mechanisms, using bioorthogonal adaptors to guide site-specificity.

Scheme 1

Scheme 1. Protein Histidine PTMs, ncAAs and α,β-Unsaturated Chemicals, and Rational Design of a Ferritin-Based aza-Michael Ligase for Site-Specific Protein Histidine Modifications
High Resolution Image
Download MS PowerPoint Slide

Although increasing electrophile concentrations (2,13) and tunable metallaphotoredox potentials (14) can help obtain higher yield, it is often at the cost of selectivity. The presence of the many interfering, more reactive, and repetitive functional groups within proteins further impedes selective formation of covalent bonds with histidine’s less nucleophilic side chain. Hence, histidine-specific modifications are often dependent on ligand- and/or sequence-directed chemistry in addition to harsh reaction conditions to achieve chemoselectivity. (15) While novel reagents have also been explored for these to be performed under relatively mild reaction conditions (16) and chemical derivatization followed by further HPLC purification may help achieve protein single-site histidine modification, (17) methodologies to achieve site-selectivity remain underexplored. With the ultimate objective to create a biocatalysis platform that could be expressed in vivo, we report the rational design of an artificial human ferritin heavy chain (FTH1)-based metalloprotein to achieve histidine-specific aza-Michael addition with α,β-unsaturated chemical moieties (Scheme 1).

Artificial metalloproteins are powerful biocatalysts for their metal-chelating properties. In fact, many of those reported are based on scaffold proteins engineered to anchor transition metals. (18) In search of a protein scaffold for chelating Cu(II), the Lewis acid that is commonly applied to catalyze aza-Michael addition, to modify proteins, we came across a L56H/R63H/E67H ferritin (Ftn) mutant (Figure S1B) that assembles only upon copper binding between the above three histidine residues alongside a native H60 (19) (Figures 1A and S1A). As an iron storage and release self-assembling protein cage, wild-type Ftn (wt-Ftn) comprises 24 FTH1 subunits with 12 C2 axes (20) (Figure S2). Utilizing the large area the C2 interfaces cover altogether, mutated FTH1 subunits were designed to assemble only upon divalent copper binding at the C2 interfaces hosting their histidine mutations. To still allow these sites and/or structurally induced nearby residues to chelate Cu(II) for catalyzing histidine modifications while allowing the ferritin cage to reserve its self-assembling property, instead of modifying all of the above-mentioned four copper-chelating histidines, we attempted to replace only one (R63) or two (R63/E67), which are noncopper-chelating, with histidine analogs 1–3 (Scheme 1) (4-thiazolyl-l-alanine, 1; 2-(5-bromothienyl)-l-alanine (BtA), 2; 3-methyl-l-histidine (MeH), 3), separately, via genetic code expansion (21) (Figure S3). These noncanonical amino acids (ncAAs) were previously reported to chelate Cu(II) in either nature or synthesized ligands. Using the MmPylRS-N346A/C348A·tRNAPyl pair (22) bioorthogonal to the Escherichia coli system, we generated six Ftn variants (Figure 1B).

Figure 1

Figure 1. (A) R63 and E67 of wt-Ftn (PDB code: 2FHA) are targeted for histidine analogs 1–3 (Scheme 1). Incorporation at the C2 interface, as shown between two FTH1 monomers (red and blue). (B) 4–12% native PAGE analyses of the six ncAA-incorporated Ftn variants, with (C) ESI-MS analyses of the six ncAA-incorporated Ftn variants and (D) their Fe(II) and Cu(II) contents measured by ICP-MS alongside those of apoferritin, equine spleen ferritin, and wt-Ftn.

High Resolution Image
Download MS PowerPoint Slide

A native PAGE analysis of the Ftn variants alongside wt-Ftn was first carried out to verify their assembly (Figure 1B). Individual molecular weights (MWs) were then confirmed by ESI-MS (Figure 1C). ICP-MS analyses were further performed to measure individual Fe(II) contents (Figure 1D), alongside commercial standards of apoferritin and equine spleen ferritin. The large disparity between Fe(II) contents in the commercial standards can be explained by Fe(II) binding only to apoferritin’s surface and not stored inside its cavity. Fe(II) content between Ftn variants and in comparison with wt-Ftn also varied significantly with signature feature amounts of 0, 24, and 48 Fe(II) ions along with the FTH1 24mers stoichiometry (Figure 1D). In the crystal structure of Ftn-1x-3 (Table S1, Figures S1C–D) without Fe(II) or Cu(II) content, additive bending hexaethylene glycol in the C2 interface binds to MeH63 (Figure S1D) and generates another potential metal binding pocket, along with the 59SHEE63 MeHEHA67E sequence that can be found in the Ftn-L56H/R63H/E67H mutant (Figures S1A, S1C).

The cryo-EM structural models (Table S2, Figures S4–S7) of Ftn-2X-2 (Figures 2A,B) and Ftn-2x-3 (Figures 2E,F) and these two Ftns with CuCl2 supplement (Figures 2C,D, 2G,H) were solved and revealed two detailed metal binding environments. Instate of binding metal, either Fe(II) or Cu(II), at the C2 interface of Ftn-2x-2, 63BtA, 67BtA, and H60 form an aromatic cluster of pi–pi interactions (Figures 2A,C). Thus, E27/E62/H65 turns to bind to Cu(II) in Ftn-2x-2-Cu(II) (Figure 2D) by replacing the sodium ion (Figure 2B) originally. In fully Fe(II)-bonded Ftn-2x-3, S59/H60/63MeH at the C2 interface (Figure 2E) and E27/E62/H65 (Figure 2F) binding sites are identified. In a comparison to the ICP-MS data and structural models of Ftn-1x-3 (Figure S1D) and Ftn-2x-3 (Figure 2E), the E67MeH mutation displays dual roles in aromatic ring stacking and metal chelating in controlling Fe(II) and Cu(II) binding. After Cu(II) supplementation, S59/H60/63MeH at the C2 interface loses the binding to Fe(II) and forms stable hydrophobic interactions (Figure 2G). The local interactions make the binding distance of E27 slightly longer, 3.1 Å, and lead to another Cu(II) binding site at E62 and E107. With the original E27/E62/H65 binding motif, Ftn-2x-3-Cu(II) forms a nonheme dicopper center (Figure 2H).

Figure 2

Figure 2. Cryo-EM structures of metal binding sites at the C2 engineered interface and monomer of (A, B) Ftn-2x-2 (PDB code 9JQB), (C, D) Ftn-2x-2-Cu(II) (PDB code: 9JQC), (E, F) Ftn-2x-3 (PDB code: 9JQD), and (G, H) Ftn-2x-3-Cu(II) (PDB code: 9JQE). Protein residues are depicted as light blue sticks, sodium ions in purple, copper in yellow, and iron in orange. Cryo-EM density maps are colored transparent gray.

High Resolution Image
Download MS PowerPoint Slide

In order to minimize interference of Fe(II) during catalysis, we selected the two variants with the lowest iron contents, Ftn-1x-3 and Ftn-2x-2 (Figure 1D), for further treatment and characterization in the catalytic property. Upon CuCl2 treatment, Cu(II) contents in the two selected variants notably increased compared with wt-Ftn, whereas there were no significant changes to their Fe(II) contents. With the structural analysis of the Cu(II) binding site at E27/E62/H65 of Ftn-2x-2-Cu(II) and Ftn-2x-3-Cu(II), these results indicated that our engineering is responsible for the impairment of Ftn’s Fe(II) binding. To examine the new metalloenzymes’ viability as aza-Michael ligases, we attempted to modify the human insulin protein with diethyl ethylidenemalonate (DEEM, 4, Scheme 1).

Out of insulin’s 51 amino acids, two are histidine (B-chain H5 and H10) (23) (Figure 3A). Being an essential medication for treating diabetes, much effort has been made to chemically modify this large peptide hormone to fine-tune its time–action profile. Though existing technologies have indeed increased its varieties considerably, most modifications target the most reactive lysine residue. Hence, establishing a biocatalysis platform capable of site-selectively modifying histidine is without doubt invaluable for further enriching the diversity of insulin analogs. Single modifications of DEEM (1x-DEEM), as represented by 186 Da shifts on MALDI-TOF-MS spectra, suggested our newly designed metalloenzymes to be carrying out modifications with specificity (Figures 3A–C and S8–S10). Conversion rates (CVRs) carried out by Ftn-1x-3 and Ftn-2x-2 with 2 equiv of DEEM were 20% and 33%, respectively, making the latter more desirable for further characterization. For Ftn-2x-2, seven other α,β-unsaturated reagents (5–11) (Scheme 1) were tested to modify insulin, yielding CVRs between 0 and 44% (Figure 4A and Figures S11–S12). Quite interestingly, with α,β-unsaturated sulfones (10, 11), the enzyme exhibited comparably higher activity yet less selectivity in modifying histidine.

Figure 3

Figure 3. MALDI-TOF-MS analyses of the insulin protein either (A) unmodified or modified with a single DEEM adduct (+186 m/z or Da) by CuCl2-treated (B) Ftn-1x-3 (CVR = 20%, turnover number (TON) = 4.0 h–1 ball–1), (C) Ftn-2x-2 (CVR = 33%, TON = 6.6 h–1 ball–1), (D) Ftn-α-1x-3 (CVR = 46%, TON = 9.2 h–1 ball–1), and (E) Ftn-α-2x-2 (CVR = ∼100%, TON = 24 h–1 ball–1).

High Resolution Image
Download MS PowerPoint Slide

Figure 4

Figure 4. (A) Yield of Ftn-2x-2’s α,β-unsaturated chemical moiety-modified insulin products; (B) Amino acid, histidine and cysteine count, and yield of Ftn-2x-2’s DEEM-modified peptide and protein products.

High Resolution Image
Download MS PowerPoint Slide

To investigate Ftn-2x-2’s substrate scope as an aza-Michael ligase, we examined its catalytic activity alongside DEEM treatment on seven additional protein substrates that ranged in length from 10 to 607 residues: a luteinizing hormone-releasing hormone analog ([d-Ala6, N-Me-Leu7]-LH-RH), the small ubiquitin-related modifier 1 (SUMO1), lysozyme, human ubiquitin conjugating enzymes UBE2 V2 and UBE2N, superfolder green fluorescent protein (sfGFP), and bovine serum albumin (BSA). Aside from UBE2 V2 hosting no histidine, again, only adducts with single modifications were observed (Figures 3C, 4B, and S19–S25). MALDI-TOF-MS/MS further confirmed site-specific DEEM modification on [d-Ala6, N-Me-Leu7]-LH-RH’s H2, whereas due to the lower yield of SUMO1–1x-DEEM, however, no DEEM modifications were revealed in its N-terminal and three histidine-containing peptides during ESI-MS/MS analyses (Figures S26–S30). Nonetheless, a carbamidomethyl modification commonly observed in in-gel digestion samples has been identified on SUMO1’s C51, indicating that its only cysteine is not DEEM-modified (Figure S31).

With these promising preliminary data, we continued to characterize DEEM’s location in Ftn-2x-2’s modified insulin products by reducing all disulfide bonds with 30 equiv of DTT (Figure 5A). In both modified and unmodified insulin, A-chains were found to remain soluble, whereas B-chains precipitated. MALDI-TOF-MS analyses further revealed two DEEM-modified species (insulin B chain-1x-DEEM and −2x-DEEM) to be present, with the second only in trace amounts, but are both located only on the insulin B-chain, where the two histidine residues (Figures 5B,C and S32).

Figure 5

Figure 5. (A) Ftn-2x-2’s DEEM-modified insulin product is treated with DTT to be digested into (B) A- and (C) B-chains, which are then separately analyzed by MALDI-TOF-MS to determine DEEM adduct’s localization. (D) MALDI-TOF-MS/MS analysis of Ftn-α-2x-2’s DEEM-modified insulin product revealed the site-selective modification to be carried out on only insulin’s B-chain H5.

High Resolution Image
Download MS PowerPoint Slide

In cells, chemo- and site-selective PTM is guided by the enzyme recognition of target sequences in protein substrates. Based on this rationale, to achieve site-selectivity and increase the catalytic efficiency of our metalloenzymes, we intend to engineer a target recognition peptide (TRP) into our Ftn variants. αCT is a 16-amino acid peptide isolated from the insulin receptor’s substrate-binding domain, and is known to build contacts with both insulin chains (24) (Figure S33). We noted that H5 of insulin B-chain is located within its N-terminal nonstructural region, whereas H10 from the same chain is located within its first α-helical region, which would be stabilized upon insulin’s interaction with αCT. Hence, we hypothesize that by utilizing αCT as a TRP, we could site-selectively target H5 for modification, taking advantage of its flexibility to further interact with Cu(II). To optimize the design of our meta-lloenzymes, we generated a series of His-tagged Ftn-αCT-fusion variants (Figures 3D,E), with the peptides fused in distinct orders (Figure S34A) and via flexible linkers of varying lengths (Figure S35A). The N-termini of wt-FTH1 subunits are known to be surface-exposed, while the C-termini are buried within the protein (Figure S2). Interestingly, native and SDS-PAGE results revealed that the fusion of αCT at FTH1’s N-terminus (α-Ftn) would impair Ftn’s assembly, making the protein insoluble in buffer, whereas the C-terminally fused variant (Ftn-α) remained soluble, assembled, and could be readily purified through Ni2+-NTA columns via its C-terminal 6xHis-tag (Figures S34B–S34D).

To then adjust the linker’s length so to understand its correlation with the 6xHis-tag’s accessibility, three constructs with linkers of diverging lengths (S(GGGGS)1–3, L1–3) were produced (Figure S35A). A native PAGE analysis confirmed all variants’ assembly (Figure S34E). Through SDS-PAGE visualization of elution profiles, we observed that among the three variants, the longer the linker, the higher the binding affinity (Figures S35B–S35D). Thus, further engineering of Ftn-α-1x-3 and Ftn-α-2x-2 variants were completed with the L3 linker. Homogenous ncAA incorporations in both variants were first corroborated via ESI-MS (Figure S36). MALDI-TOF-MS analyses then confirmed both variants to yield only single DEEM modification peaks and near 26% and 57% CVR increases, respectively, as compared to their respective counterparts without αCT-fusions (Figures 3B,E and S37–S38). These increases indicated our design of a TRP to be successful in increasing the ligases’ catalytic efficiency. Moreover, one equivalent of insulin and two equiv of DEEM, the Michael acceptor, were used in enzymatic reactions ([insulin]:[DEEM]:[Ftn-a-2x-2] = 1:2:0.04) to achieve near quantitative conversion by Ftn-α-2x-2, although it is with a lower TON. Ftn-α-2x-2’s modified products were further digested by endoproteinase Glu-C and analyzed by MALDI-TOF-MS/MS, demonstrating DEEM modifications to be carried out site-selectively on only H5 of insulin’s B-chain, as we intended for (Figure 5D). DLS and FEG-TEM analyses were additionally carried out to confirm Ftn-α-2x-2’s similar morphology with wt-Ftn in terms of size and shape when mixed with insulin (Figures S39–S40). Combining these results, we report Ftn-α-2x-2 to be an effective aza-Michael ligase that site-specifically targets H5 of the insulin B-chain. Further research removing Fe(II) from Ftn variants prior to CuCl2 treatment will be reported in due course to elucidate factors that contribute to the catalysis with new Cu(II) binding site.

In conclusion, we have reported a biocatalysis platform inspired by native PTM mechanisms that can carry out site-selective histidine modifications on proteins. Complementing existing methods for chemoselective histidine modification, we envision this platform to empower us to produce a wider array of homogeneous bioconjugates. This human protein-based platform further sets the stage for developing novel enzyme therapies, for instance, to modify native insulin of diabetic patients by fine-tuning its action profile in vivo.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c14446.

  • List of abbreviations, Ftn variants, peptide/protein sequences, plasmids and primers, Materials and Method, Supplementary Tables S1–S4, and Supplementary Figures S1–S35, including results from MS, X-ray crystallography, Cryo-EM, PAGE, DLS and TEM analyses (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
  • Authors
    • Jo-Chu Tsou - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    • Chun-Ju Tsou - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;  Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
    • Chun-Hsiung Wang - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    • An-Li A. Ko - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    • Yi-Hui Wang - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;  Orcidhttps://orcid.org/0009-0002-2616-4129
    • Huan-Hsuan Liang - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;  Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
    • Jia-Cheng Sun - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    • Kai-Fa Huang - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
    • Tzu-Ping Ko - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;  Orcidhttps://orcid.org/0000-0003-1794-2638
    • Shu-Yu Lin - Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
  • Author Contributions

    J.-C.T., C.-J.T., C.-H.W., and A.-L.A.K. have contributed equally to this work and share first authorship.

  • Funding

    The authors declare financial support was received for the research, authorship, and publication of this article. This research was supported by Academia Sinica, the Ministry of Science and Technology (MOST 107-2113-M-001-025-MY3), the National Science and Technology Council (NSTC 113-2113-M-001-006 and NSTC 112-2113-M-001-015), and the National Synchrotron Radiation Research Center (NSRRC) (2023-1-235-1 and 2024-1-223-1).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

We thank Dr. Han-Kai Jiang for assistance with in-gel digestion, Mr. Mu-Lung Jian for ferritin preparation, Dr. Meng-Ru Ho for DLS analyses, and Ms. Cheng-Hsilin Hsieh for MALDI-TOF-MS support. ESI-MS data were acquired at the AS Common Mass Spectrometry Facility, DLS at the Biophysics Core Facility, and ICP-MS at the Genomics Research Center, AS. We also acknowledge the AS Biological Electron Microscopy Core Facility, Academia Sinica Cryo-EM Facility, Academia Sinica Grid-computing Center, IBC Protein X-ray Crystallography Facility, and NSRRC beamlines TLS-15A1, TPS-07A1, and TPS-05A1.

References

Click to copy section linkSection link copied!

This article references 24 other publications.

  1. 1
    Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J., Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem., Int. Ed. Engl. 2005, 44 (45), 7342– 7372,  DOI: 10.1002/anie.200501023
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Taylor, R. J.; Geeson, M. B.; Journeaux, T.; Bernardes, G. J. L. Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation. J. Am. Chem. Soc. 2022, 144 (32), 14404– 14419,  DOI: 10.1021/jacs.2c00129
    Google Scholar
    2
    Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation
    Taylor, Ross J.; Geeson, Michael B.; Journeaux, Toby; Bernardes, Goncalo J. L.
    Journal of the American Chemical Society (2022), 144 (32), 14404-14419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biol. activity. This perspective focuses on chem. and enzymic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chem. modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges assocd. with using chem. and enzymic approaches are highlighted and areas requiring further development are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvFKit7nE&md5=ac47fd3f80fbcc053c233c48c6e59e5e
  3. 3
    Walsh, G. Post-translational modifications of protein biopharmaceuticals. Drug Discov Today 2010, 15 (17–18), 773– 780,  DOI: 10.1016/j.drudis.2010.06.009
    Google Scholar
    There is no corresponding record for this reference.
    Bell, E. L.; Finnigan, W.; France, S. P.; Green, A. P.; Hayes, M. A.; Hepworth, L. J.; Lovelock, S. L.; Niikura, H.; Osuna, S.; Romero, E. Biocatalysis. Nature Reviews Methods Primers 2021,  DOI: 10.1038/s43586-021-00044-z
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Walsh, G.; Jefferis, R. Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 2006, 24 (10), 1241– 1252,  DOI: 10.1038/nbt1252
    Google Scholar
    4
    Post-translational modifications in the context of therapeutic proteins
    Walsh, Gary; Jefferis, Roy
    Nature Biotechnology (2006), 24 (10), 1241-1252CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    A review. The majority of protein-based biopharmaceuticals approved or in clin. trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, addnl. PTMs, including carboxylation, hydroxylation, sulfation, and amidation, are characteristic of some products. The relation between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structure-function relations will facilitate the development of 2nd-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVGgsrnK&md5=3376c8170602376c6fe0505c32533828
  5. 5
    Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Engineering the third wave of biocatalysis. Nature 2012, 485 (7397), 185– 194,  DOI: 10.1038/nature11117
    Google Scholar
    5
    Engineering the third wave of biocatalysis
    Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K.
    Nature (London, United Kingdom) (2012), 485 (7397), 185-194CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review. Over the past ten years, scientific and technol. advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chem. synthesis, both in the lab. and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chems. to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmvVeqsLk%253D&md5=5f20c530c25ea886f5f5d33dbea0075a
  6. 6
    Petrovic, D. M.; Bissaro, B.; Chylenski, P.; Skaugen, M.; Sorlie, M.; Jensen, M. S.; Aachmann, F. L.; Courtade, G.; Varnai, A.; Eijsink, V. G. H. Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation. Protein Sci. 2018, 27 (9), 1636– 1650,  DOI: 10.1002/pro.3451
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Macek, B.; Forchhammer, K.; Hardouin, J.; Weber-Ban, E.; Grangeasse, C.; Mijakovic, I. Protein post-translational modifications in bacteria. Nat. Rev. Microbiol 2019, 17 (11), 651– 664,  DOI: 10.1038/s41579-019-0243-0
    Google Scholar
    7
    Protein post-translational modifications in bacteria
    Macek, Boris; Forchhammer, Karl; Hardouin, Julie; Weber-Ban, Eilika; Grangeasse, Christophe; Mijakovic, Ivan
    Nature Reviews Microbiology (2019), 17 (11), 651-664CODEN: NRMACK; ISSN:1740-1526. (Nature Research)
    A review. Over the past decade the no. and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein modifications occur in a relatively low no. of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional anal. is particularly challenging. The no. of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metab., the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiol. and open new avenues for treatment of infectious diseases.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWnsbfK&md5=099ac24cd64ce48e27cf4499a19c059b
    Zhong, Q.; Xiao, X.; Qiu, Y.; Xu, Z.; Chen, C.; Chong, B.; Zhao, X.; Hai, S.; Li, S.; An, Z.; Dai, L. Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications. MedComm 2023,  DOI: 10.1002/mco2.261
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Mathis, L. C.; Barrios, M. A. Histidine phosphorylation in metalloprotein binding sites. Journal of Inorganic Biochemistry 2021, 225, 111606  DOI: 10.1016/j.jinorgbio.2021.111606
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Hayashi, T.; Daitoku, H.; Uetake, T.; Kako, K.; Fukamizu, A. Histidine Nτ-methylation identified as a new posttranslational modification in histone H2A at His-82 and H3 at His-39. J. Biol. Chem. 2023, 299 (9), 105131  DOI: 10.1016/j.jbc.2023.105131
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Tekaia, F.; Yeramian, E.; Dujon, B. Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene 2002, 297 (1–2), 51– 60,  DOI: 10.1016/S0378-1119(02)00871-5
    Google Scholar
    There is no corresponding record for this reference.
    Brune, D.; Andrade-Navarro, M. A.; Mier, P. Proteome-wide comparison between the amino acid composition of domains and linkers. BMC Res. Notes 2018, 11 (1), 117,  DOI: 10.1186/s13104-018-3221-0
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Burke, A. J.; Lovelock, S. L.; Frese, A.; Crawshaw, R.; Ortmayer, M.; Dunstan, M.; Levy, C.; Green, A. P. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature 2019, 570 (7760), 219– 223,  DOI: 10.1038/s41586-019-1262-8
    Google Scholar
    11
    Design and evolution of an enzyme with a non-canonical organocatalytic mechanism
    Burke, Ashleigh J.; Lovelock, Sarah L.; Frese, Amina; Crawshaw, Rebecca; Ortmayer, Mary; Dunstan, Mark; Levy, Colin; Green, Anthony P.
    Nature (London, United Kingdom) (2019), 570 (7760), 219-223CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The combination of computational design and lab. evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-mol. organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in soln. Crystallog. snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chem. transformations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFWqtLfN&md5=66888acccecd387d49e7ae47fef06d83
  12. 12
    Sharma, V.; Wang, Y. S.; Liu, W. R. Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics. ACS Chem. Biol. 2016, 11 (12), 3305– 3309,  DOI: 10.1021/acschembio.6b00940
    Google Scholar
    12
    Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics
    Sharma, Vangmayee; Wang, Yane-Shih; Liu, Wenshe R.
    ACS Chemical Biology (2016), 11 (12), 3305-3309CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine can be genetically encoded using an engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2ju7vI&md5=29f5b55e8f74bf2e3577c510ce1c31d1
  13. 13
    Boutureira, O.; Bernardes, G. J. Advances in chemical protein modification. Chem. Rev. 2015, 115 (5), 2174– 2195,  DOI: 10.1021/cr500399p
    Google Scholar
    13
    Advances in Chemical Protein Modification
    Boutureira, Omar; Bernardes, Goncalo J. L.
    Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Transition metal-free and -mediated approaches are covered.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nu7k%253D&md5=d21a5407a59a7e12346c16e5db75ab91
  14. 14
    King, T. A.; Mandrup Kandemir, J.; Walsh, S. J.; Spring, D. R. Photocatalytic methods for amino acid modification. Chem. Soc. Rev. 2021, 50 (1), 39– 57,  DOI: 10.1039/D0CS00344A
    Google Scholar
    14
    Photocatalytic methods for amino acid modification
    King, Thomas A.; Mandrup Kandemir, Jiyan; Walsh, Stephen J.; Spring, David R.
    Chemical Society Reviews (2021), 50 (1), 39-57CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Amino acid modification plays an important role across several fields, including synthetic org. chem., materials science, targeted drug delivery and the probing of biol. function. Although a myriad of methods now exist for the modification of peptides or proteins, many of these target a handful of the most reactive proteinogenic amino acids. Photocatalysis has recently emerged as a mild approach for amino acid modification, generating a sizable toolbox of reactions capable of modifying almost all of the canonical amino acids. These reactions are characterised by their mild, physiol. compatible conditions, greatly enhancing their usefulness for amino acid modification. This review aims to introduce the field of photocatalytic amino acid modification and discusses the most recent advances.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit12ktbnL&md5=8d310120eb6697790104b242e51b0609
    Bloom, S.; Liu, C.; Kolmel, D. K.; Qiao, J. X.; Zhang, Y.; Poss, M. A.; Ewing, W. R.; MacMillan, D. W. C. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 2018, 10 (2), 205– 211,  DOI: 10.1038/nchem.2888
    Google Scholar
    14
    Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials
    Bloom, Steven; Liu, Chun; Kolmel, Dominik K.; Qiao, Jennifer X.; Zhang, Yong; Poss, Michael A.; Ewing, William R.; MacMillan, David W. C.
    Nature Chemistry (2018), 10 (2), 205-211CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    The advent of antibody-drug conjugates as pharmaceuticals has fueled 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 soln. to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, the authors explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, the authors demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidn. potentials between internal vs. C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidn. potential-gated technol. is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodol. provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWgsrjJ&md5=a5f8b7f701329a625221c77280243746
  15. 15
    Vohidov, F.; Coughlin, J. M.; Ball, Z. T. Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains. Angew. Chem., Int. Ed. Engl. 2015, 54 (15), 4587– 4591,  DOI: 10.1002/anie.201411745
    Google Scholar
    There is no corresponding record for this reference.
    Peciak, K.; Laurine, E.; Tommasi, R.; Choi, J. W.; Brocchini, S. Site-selective protein conjugation at histidine. Chemical Science 2019, 10 (2), 427– 439,  DOI: 10.1039/C8SC03355B
    Google Scholar
    There is no corresponding record for this reference.
    Nakane, K.; Sato, S.; Niwa, T.; Tsushima, M.; Tomoshige, S.; Taguchi, H.; Ishikawa, M.; Nakamura, H. Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen. J. Am. Chem. Soc. 2021, 143 (20), 7726– 7731,  DOI: 10.1021/jacs.1c01626
    Google Scholar
    15
    Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen
    Nakane, Keita; Sato, Shinichi; Niwa, Tatsuya; Tsushima, Michihiko; Tomoshige, Shusuke; Taguchi, Hideki; Ishikawa, Minoru; Nakamura, Hiroyuki
    Journal of the American Chemical Society (2021), 143 (20), 7726-7731CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small mol., 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liq. chromatog.-mass spectrometry anal. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpsFehs7Y%253D&md5=d9bac378e73ba9c624abd20cb6489a23
    Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi, I. One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity. J. Am. Chem. Soc. 2006, 128 (10), 3273– 3280,  DOI: 10.1021/ja057926x
    Google Scholar
    15
    One-Pot and Sequential Organic Chemistry on an Enzyme Surface to Tether a Fluorescent Probe at the Proximity of the Active Site with Restoring Enzyme Activity
    Takaoka, Yousuke; Tsutsumi, Hiroshi; Kasagi, Noriyuki; Nakata, Eiji; Hamachi, Itaru
    Journal of the American Chemical Society (2006), 128 (10), 3273-3280CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A new and simple method to tether a functional mol. at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivs., so that it restored the enzymic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping expt. revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chem., the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1CitbY%253D&md5=959d5fc717b79240bad70c3d7e12feef
  16. 16
    Labroo, V. M.; Labroo, R. B.; Cohen, L. A. Direct Photochemical Trifluoromethylation of Histidine-Containing Peptides. Tetrahedron Lett. 1990, 31 (40), 5705– 5708,  DOI: 10.1016/S0040-4039(00)97937-1
    Google Scholar
    16
    Direct photochemical trifluoromethylation of histidine-containing peptides
    Labroo, V. M.; Labroo, R. B.; Cohen, L. A.
    Tetrahedron Letters (1990), 31 (40), 5705-8CODEN: TELEAY; ISSN:0040-4039.
    Photochem. trifluoromethylation of the imidazole ring of histidine in the tripeptide Glp-His-Pro-NH2 has been achieved to furnish a mixt. of imidazole trifluoromethylated isomers, which have been sepd. by reverse-phase HPLC and characterized.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXisVGruw%253D%253D&md5=972dbf43e25be716d0bddbe5d1f1f930
    Wan, C.; Wang, Y.; Lian, C.; Chang, Q.; An, Y.; Chen, J.; Sun, J.; Hou, Z.; Yang, D.; Guo, X. Histidine-specific bioconjugation via visible-light-promoted thioacetal activation. Chem. Sci. 2022, 13 (28), 8289– 8296,  DOI: 10.1039/D2SC02353A
    Google Scholar
    16
    Histidine-specific bioconjugation via visible-light-promoted thioacetal activation
    Wan, Chuan; Wang, Yuena; Lian, Chenshan; Chang, Qi; An, Yuhao; Chen, Jiean; Sun, Jinming; Hou, Zhanfeng; Yang, Dongyan; Guo, Xiaochun; Yin, Feng; Wang, Rui; Li, Zigang
    Chemical Science (2022), 13 (28), 8289-8296CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addn. to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine contg. proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhs12ls7jI&md5=30b9b2abd14a803c14a2c241ccf04b95
    Chen, X.; Ye, F.; Luo, X.; Liu, X.; Zhao, J.; Wang, S.; Zhou, Q.; Chen, G.; Wang, P. Histidine-Specific Peptide Modification via Visible-Light-Promoted C-H Alkylation. J. Am. Chem. Soc. 2019, 141 (45), 18230– 18237,  DOI: 10.1021/jacs.9b09127
    Google Scholar
    16
    Histidine-specific peptide modification via visible-light-promoted C-H alkylation
    Chen, Xiaoping; Ye, Farong; Luo, Xiaosheng; Liu, Xueyi; Zhao, Jie; Wang, Siyao; Zhou, Qingqing; Chen, Gong; Wang, Ping
    Journal of the American Chemical Society (2019), 141 (45), 18230-18237CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Histidine (His) carries a unique heteroarom. imidazole side chain and plays irreplaceable functional roles in peptides and proteins. Existing strategies for site-selective histidine modification predominantly rely on the N-substitution reactions of the moderately nucleophilic imidazole group, which inherently suffers from the interferences from lysine and cysteine residues. Chemoselective modification of histidine remains one of the most difficult challenges in peptide chem. Herein, we report peptide modification via radical-mediated chemoselective C-H alkylation of histidine using C4-alkyl-1,4-dihydropyridine (DHP) reagents under visible-light-promoted conditions. The method exploits the electrophilic reactivity of the imidazole ring via a Minisci-type reaction pathway. This method exhibits an exceptionally broad scope for both peptides and DHP alkylation reagents. Its utility has been demonstrated in a series of important peptide drugs, complex natural products, and a small protein. Distinct from N-substitution reactions, the unsubstituted nitrogen groups of the modified imidazole ring are conserved in the C-H alkylated products.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvF2kurvE&md5=3625d153fe10e6ad4fde7786097f9dd0
    Jia, S.; He, D.; Chang, C. J. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. J. Am. Chem. Soc. 2019, 141 (18), 7294– 7301,  DOI: 10.1021/jacs.8b11912
    Google Scholar
    16
    Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation
    Jia, Shang; He, Dan; Chang, Christopher J.
    Journal of the American Chemical Society (2019), 141 (18), 7294-7301CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. The authors now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic posttranslational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddn. (CuAAC) chem. The authors establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purifn., as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chem.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnvFCgsL8%253D&md5=57725240e216d1c915749ed964081b13
    Uchida, K.; Stadtman, E. R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544– 4548,  DOI: 10.1073/pnas.89.10.4544
    Google Scholar
    16
    Modification of histidine residues in proteins by reaction with 4-hydroxynonenal
    Uchida, Koji; Stadtman, E. R.
    Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (10), 4544-8CODEN: PNASA6; ISSN:0027-8424.
    Histidine residues in proteins are major targets for reaction with the lipid peroxidn. product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivs. upon subsequent treatment with NaB[3H]H4. Amino acid anal. of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stoichiometric formation of 3H-labeled amino acid hydrolyzates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the prodn. of two compds. Upon acid hydrolysis, both derivs. yielded stoichiometric amts. of histidine. However, after redn. with NaBH4, acid hydrolysis led to a mixt. of amino acid derivs. [presumably, isomeric forms of Nπ(Nτ)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addn. of the imidazole nitrogen atom of histidine to the α,β-unsatd. bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be detd.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVeqtL4%253D&md5=e319c0a1200aa40784b00fde9bdd7cd6
  17. 17
    Joshi, P. N.; Rai, V. Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification. Chem. Commun. (Camb) 2019, 55 (8), 1100– 1103,  DOI: 10.1039/C8CC08733D
    Google Scholar
    There is no corresponding record for this reference.
    Rawale, D. G.; Thakur, K.; Sreekumar, P.; Sajeev, T. K.; Ramesh, A.; Adusumalli, S. R.; Mishra, R. K.; Rai, V. Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins. Chem. Sci. 2021, 12 (19), 6732– 6736,  DOI: 10.1039/D1SC00335F
    Google Scholar
    17
    Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins
    Rawale, Dattatraya Gautam; Thakur, Kalyani; Sreekumar, Pranav; T. K., Sajeev; A., Ramesh; Adusumalli, Srinivasa Rao; Mishra, Ram Kumar; Rai, Vishal
    Chemical Science (2021), 12 (19), 6732-6736CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create a unique chem. space to enable site-selectivity for histidine and aspartic acid modifications overcoming the pre-requisite of chemoselectivity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotlygs7w%253D&md5=a13bce7fd4e21e1865e6b7823ecf164c
  18. 18
    Drienovska, I.; Rioz-Martinez, A.; Draksharapu, A.; Roelfes, G. Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids. Chem. Sci. 2015, 6 (1), 770– 776,  DOI: 10.1039/C4SC01525H
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Huard, D. J.; Kane, K. M.; Tezcan, F. A. Re-engineering protein interfaces yields copper-inducible ferritin cage assembly. Nat. Chem. Biol. 2013, 9 (3), 169,  DOI: 10.1038/nchembio.1163
    Google Scholar
    19
    Re-engineering protein interfaces yields copper-inducible ferritin cage assembly
    Huard, Dustin J. E.; Kane, Kathleen M.; Tezcan, F. Akif
    Nature Chemical Biology (2013), 9 (3), 169-176CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    The ability to chem. control protein-protein interactions would allow the interrogation of dynamic cellular processes and lead to a better understanding and exploitation of self-assembling protein architectures. Here we introduce a new engineering strategy - reverse metal-templated interface redesign (rMeTIR) - that transforms a natural protein-protein interface into one that only engages in selective response to a metal ion. We have applied rMeTIR to render the self-assembly of the cage-like protein ferritin controllable by divalent copper binding, which has allowed the study of the structure and stability of the isolated ferritin monomer, the demonstration of the primary role of conserved hydrogen-bonding interactions in providing geometric specificity for cage assembly and the uniform chem. modification of the cage interior under physiol. conditions. Notably, copper acts as a structural template for ferritin assembly in a manner that is highly reminiscent of RNA sequences that template virus capsid formation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFKqsL8%253D&md5=e52b99e1124df8ac2892b12e70f2e655
  20. 20
    Hempstead, P. D.; Yewdall, S. J.; Fernie, A. R.; Lawson, D. M.; Artymiuk, P. J.; Rice, D. W.; Ford, G. C.; Harrison, P. M. Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 1997, 268 (2), 424– 448,  DOI: 10.1006/jmbi.1997.0970
    Google Scholar
    20
    Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution
    Hempstead, Paul D.; Yewdall, Stephen J.; Fernie, Alisdair R.; Lawson, David M.; Artymiuk, Peter J.; Rice, David W.; Ford, Geoffrey C.; Harrison, Pauline M.
    Journal of Molecular Biology (1997), 268 (2), 424-448CODEN: JMOBAK; ISSN:0022-2836. (Academic)
    Mammalian ferritins are 24-mers assembled from two types of polypeptide chain which provide the mol. with different functions. Heavy (H) chains catalyze the first step in iron storage, the oxidn. of iron(II). Light (L) chains promote the nucleation of the mineral ferrihydrite enabling storage of iron(III) inside the protein shell. We report here the comparison of the three-dimensional structures of recombinant human H chain (HuHF) and horse L chain (HoLF) ferritin homopolymers, which have been refined at 1.9 Å resoln. There is 53% sequence identity between these mols., and the two structures are very similar, the H and L subunit α-carbons superposing to within 0.5 Å rms deviation with 41 water mols. in common. Nevertheless, there are significant important differences which can be related to differences in function. In particular, the centers of the four-helix bundles contain distinctive groups of hydrophilic residues which have been assocd. with ferroxidase activity in H chains and enhanced stability in L chains. L chains contain a group of glutamates assocd. with mineralization within the iron storage cavity of the protein.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjsValuro%253D&md5=ad1c56bf687a2c402329f517c58c2eed
  21. 21
    Xiao, H.; Peters, F. B.; Yang, P. Y.; Reed, S.; Chittuluru, J. R.; Schultz, P. G. Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase. ACS Chem. Biol. 2014, 9 (5), 1092– 1096,  DOI: 10.1021/cb500032c
    Google Scholar
    21
    Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase
    Xiao, Han; Peters, Francis B.; Yang, Peng-Yu; Reed, Sean; Chittuluru, Johnathan R.; Schultz, Peter G.
    ACS Chemical Biology (2014), 9 (5), 1092-1096CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogs in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase (PylRS) mutant was able to site-specifically incorporate 3-methylhistidine, 3-pyridylalanine, 2-furylalanine, and 3-(2-thienyl)alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogs created mutant proteins with distinct spectral properties. This work further expands the structural and chem. diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVOqt70%253D&md5=d94387a5111558bb29264e40f2572570
  22. 22
    Wang, Y.-S.; Fang, X.; Wallace, A. L.; Wu, B.; Liu, W. R. A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. J. Am. Chem. Soc. 2012, 134 (6), 2950– 2953,  DOI: 10.1021/ja211972x
    Google Scholar
    22
    A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a Broad Substrate Spectrum
    Wang, Yane-Shih; Fang, Xinqiang; Wallace, Ashley L.; Wu, Bo; Liu, Wenshe R.
    Journal of the American Chemical Society (2012), 134 (6), 2950-2953CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Together with tRNACUAPyl, a rationally designed pyrrolysyl-tRNA synthetase (PylRS) mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivs. with large para substituents into superfolder green fluorescent protein (sfGFP) at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivs. using engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pairs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVyktLg%253D&md5=d5f9189997497a11a60b743c0020dda3
  23. 23
    Zaykov, A. N.; Mayer, J. P.; DiMarchi, R. D. Pursuit of a perfect insulin. Nat. Rev. Drug Discov 2016, 15 (6), 425– 439,  DOI: 10.1038/nrd.2015.36
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Menting, J. G.; Whittaker, J.; Margetts, M. B.; Whittaker, L. J.; Kong, G. K. W.; Smith, B. J.; Watson, C. J.; Záková, L.; Kletvíková, E.; Jirácek, J. How insulin engages its primary binding site on the insulin receptor. Nature 2013, 493 (7431), 241– U276,  DOI: 10.1038/nature11781
    Google Scholar
    24
    How insulin engages its primary binding site on the insulin receptor
    Menting, John G.; Whittaker, Jonathan; Margetts, Mai B.; Whittaker, Linda J.; Kong, Geoffrey K.-W.; Smith, Brian J.; Watson, Christopher J.; Zakova, Lenka; Kletvikova, Emilia; Jiracek, Jiri; Chan, Shu Jin; Steiner, Donald F.; Dodson, Guy G.; Brzozowski, Andrzej M.; Weiss, Michael A.; Ward, Colin W.; Lawrence, Michael C.
    Nature (London, United Kingdom) (2013), 493 (7431), 241-245CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Insulin receptor signaling has a central role in mammalian biol., regulating cellular metab., growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signaling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titrn. calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochem. data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXls1yquw%253D%253D&md5=4e4b1abc994ed5944becebe53034d840

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Download PDF
Go to Journal of the American Chemical Society
Get e-Alerts
Get e-Alerts

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2024, 146, 49, 33309–33315
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.4c14446
Published November 5, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Article Views

2865

Altmetric

-

Citations

-
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Scheme 1

    Scheme 1. Protein Histidine PTMs, ncAAs and α,β-Unsaturated Chemicals, and Rational Design of a Ferritin-Based aza-Michael Ligase for Site-Specific Protein Histidine Modifications
    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. (A) R63 and E67 of wt-Ftn (PDB code: 2FHA) are targeted for histidine analogs 1–3 (Scheme 1). Incorporation at the C2 interface, as shown between two FTH1 monomers (red and blue). (B) 4–12% native PAGE analyses of the six ncAA-incorporated Ftn variants, with (C) ESI-MS analyses of the six ncAA-incorporated Ftn variants and (D) their Fe(II) and Cu(II) contents measured by ICP-MS alongside those of apoferritin, equine spleen ferritin, and wt-Ftn.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Cryo-EM structures of metal binding sites at the C2 engineered interface and monomer of (A, B) Ftn-2x-2 (PDB code 9JQB), (C, D) Ftn-2x-2-Cu(II) (PDB code: 9JQC), (E, F) Ftn-2x-3 (PDB code: 9JQD), and (G, H) Ftn-2x-3-Cu(II) (PDB code: 9JQE). Protein residues are depicted as light blue sticks, sodium ions in purple, copper in yellow, and iron in orange. Cryo-EM density maps are colored transparent gray.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. MALDI-TOF-MS analyses of the insulin protein either (A) unmodified or modified with a single DEEM adduct (+186 m/z or Da) by CuCl2-treated (B) Ftn-1x-3 (CVR = 20%, turnover number (TON) = 4.0 h–1 ball–1), (C) Ftn-2x-2 (CVR = 33%, TON = 6.6 h–1 ball–1), (D) Ftn-α-1x-3 (CVR = 46%, TON = 9.2 h–1 ball–1), and (E) Ftn-α-2x-2 (CVR = ∼100%, TON = 24 h–1 ball–1).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. (A) Yield of Ftn-2x-2’s α,β-unsaturated chemical moiety-modified insulin products; (B) Amino acid, histidine and cysteine count, and yield of Ftn-2x-2’s DEEM-modified peptide and protein products.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. (A) Ftn-2x-2’s DEEM-modified insulin product is treated with DTT to be digested into (B) A- and (C) B-chains, which are then separately analyzed by MALDI-TOF-MS to determine DEEM adduct’s localization. (D) MALDI-TOF-MS/MS analysis of Ftn-α-2x-2’s DEEM-modified insulin product revealed the site-selective modification to be carried out on only insulin’s B-chain H5.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 24 other publications.

    1. 1
      Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J., Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem., Int. Ed. Engl. 2005, 44 (45), 7342– 7372,  DOI: 10.1002/anie.200501023
      There is no corresponding record for this reference.
    2. 2
      Taylor, R. J.; Geeson, M. B.; Journeaux, T.; Bernardes, G. J. L. Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation. J. Am. Chem. Soc. 2022, 144 (32), 14404– 14419,  DOI: 10.1021/jacs.2c00129
      2
      Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation
      Taylor, Ross J.; Geeson, Michael B.; Journeaux, Toby; Bernardes, Goncalo J. L.
      Journal of the American Chemical Society (2022), 144 (32), 14404-14419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A review. Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biol. activity. This perspective focuses on chem. and enzymic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chem. modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges assocd. with using chem. and enzymic approaches are highlighted and areas requiring further development are discussed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvFKit7nE&md5=ac47fd3f80fbcc053c233c48c6e59e5e
    3. 3
      Walsh, G. Post-translational modifications of protein biopharmaceuticals. Drug Discov Today 2010, 15 (17–18), 773– 780,  DOI: 10.1016/j.drudis.2010.06.009
      There is no corresponding record for this reference.
      Bell, E. L.; Finnigan, W.; France, S. P.; Green, A. P.; Hayes, M. A.; Hepworth, L. J.; Lovelock, S. L.; Niikura, H.; Osuna, S.; Romero, E. Biocatalysis. Nature Reviews Methods Primers 2021,  DOI: 10.1038/s43586-021-00044-z
      There is no corresponding record for this reference.
    4. 4
      Walsh, G.; Jefferis, R. Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 2006, 24 (10), 1241– 1252,  DOI: 10.1038/nbt1252
      4
      Post-translational modifications in the context of therapeutic proteins
      Walsh, Gary; Jefferis, Roy
      Nature Biotechnology (2006), 24 (10), 1241-1252CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      A review. The majority of protein-based biopharmaceuticals approved or in clin. trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, addnl. PTMs, including carboxylation, hydroxylation, sulfation, and amidation, are characteristic of some products. The relation between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structure-function relations will facilitate the development of 2nd-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVGgsrnK&md5=3376c8170602376c6fe0505c32533828
    5. 5
      Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Engineering the third wave of biocatalysis. Nature 2012, 485 (7397), 185– 194,  DOI: 10.1038/nature11117
      5
      Engineering the third wave of biocatalysis
      Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K.
      Nature (London, United Kingdom) (2012), 485 (7397), 185-194CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review. Over the past ten years, scientific and technol. advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chem. synthesis, both in the lab. and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chems. to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmvVeqsLk%253D&md5=5f20c530c25ea886f5f5d33dbea0075a
    6. 6
      Petrovic, D. M.; Bissaro, B.; Chylenski, P.; Skaugen, M.; Sorlie, M.; Jensen, M. S.; Aachmann, F. L.; Courtade, G.; Varnai, A.; Eijsink, V. G. H. Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation. Protein Sci. 2018, 27 (9), 1636– 1650,  DOI: 10.1002/pro.3451
      There is no corresponding record for this reference.
    7. 7
      Macek, B.; Forchhammer, K.; Hardouin, J.; Weber-Ban, E.; Grangeasse, C.; Mijakovic, I. Protein post-translational modifications in bacteria. Nat. Rev. Microbiol 2019, 17 (11), 651– 664,  DOI: 10.1038/s41579-019-0243-0
      7
      Protein post-translational modifications in bacteria
      Macek, Boris; Forchhammer, Karl; Hardouin, Julie; Weber-Ban, Eilika; Grangeasse, Christophe; Mijakovic, Ivan
      Nature Reviews Microbiology (2019), 17 (11), 651-664CODEN: NRMACK; ISSN:1740-1526. (Nature Research)
      A review. Over the past decade the no. and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein modifications occur in a relatively low no. of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional anal. is particularly challenging. The no. of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metab., the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiol. and open new avenues for treatment of infectious diseases.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWnsbfK&md5=099ac24cd64ce48e27cf4499a19c059b
      Zhong, Q.; Xiao, X.; Qiu, Y.; Xu, Z.; Chen, C.; Chong, B.; Zhao, X.; Hai, S.; Li, S.; An, Z.; Dai, L. Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications. MedComm 2023,  DOI: 10.1002/mco2.261
      There is no corresponding record for this reference.
    8. 8
      Mathis, L. C.; Barrios, M. A. Histidine phosphorylation in metalloprotein binding sites. Journal of Inorganic Biochemistry 2021, 225, 111606  DOI: 10.1016/j.jinorgbio.2021.111606
      There is no corresponding record for this reference.
    9. 9
      Hayashi, T.; Daitoku, H.; Uetake, T.; Kako, K.; Fukamizu, A. Histidine Nτ-methylation identified as a new posttranslational modification in histone H2A at His-82 and H3 at His-39. J. Biol. Chem. 2023, 299 (9), 105131  DOI: 10.1016/j.jbc.2023.105131
      There is no corresponding record for this reference.
    10. 10
      Tekaia, F.; Yeramian, E.; Dujon, B. Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene 2002, 297 (1–2), 51– 60,  DOI: 10.1016/S0378-1119(02)00871-5
      There is no corresponding record for this reference.
      Brune, D.; Andrade-Navarro, M. A.; Mier, P. Proteome-wide comparison between the amino acid composition of domains and linkers. BMC Res. Notes 2018, 11 (1), 117,  DOI: 10.1186/s13104-018-3221-0
      There is no corresponding record for this reference.
    11. 11
      Burke, A. J.; Lovelock, S. L.; Frese, A.; Crawshaw, R.; Ortmayer, M.; Dunstan, M.; Levy, C.; Green, A. P. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature 2019, 570 (7760), 219– 223,  DOI: 10.1038/s41586-019-1262-8
      11
      Design and evolution of an enzyme with a non-canonical organocatalytic mechanism
      Burke, Ashleigh J.; Lovelock, Sarah L.; Frese, Amina; Crawshaw, Rebecca; Ortmayer, Mary; Dunstan, Mark; Levy, Colin; Green, Anthony P.
      Nature (London, United Kingdom) (2019), 570 (7760), 219-223CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      The combination of computational design and lab. evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-mol. organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in soln. Crystallog. snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chem. transformations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFWqtLfN&md5=66888acccecd387d49e7ae47fef06d83
    12. 12
      Sharma, V.; Wang, Y. S.; Liu, W. R. Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics. ACS Chem. Biol. 2016, 11 (12), 3305– 3309,  DOI: 10.1021/acschembio.6b00940
      12
      Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics
      Sharma, Vangmayee; Wang, Yane-Shih; Liu, Wenshe R.
      ACS Chemical Biology (2016), 11 (12), 3305-3309CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine can be genetically encoded using an engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2ju7vI&md5=29f5b55e8f74bf2e3577c510ce1c31d1
    13. 13
      Boutureira, O.; Bernardes, G. J. Advances in chemical protein modification. Chem. Rev. 2015, 115 (5), 2174– 2195,  DOI: 10.1021/cr500399p
      13
      Advances in Chemical Protein Modification
      Boutureira, Omar; Bernardes, Goncalo J. L.
      Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Transition metal-free and -mediated approaches are covered.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nu7k%253D&md5=d21a5407a59a7e12346c16e5db75ab91
    14. 14
      King, T. A.; Mandrup Kandemir, J.; Walsh, S. J.; Spring, D. R. Photocatalytic methods for amino acid modification. Chem. Soc. Rev. 2021, 50 (1), 39– 57,  DOI: 10.1039/D0CS00344A
      14
      Photocatalytic methods for amino acid modification
      King, Thomas A.; Mandrup Kandemir, Jiyan; Walsh, Stephen J.; Spring, David R.
      Chemical Society Reviews (2021), 50 (1), 39-57CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Amino acid modification plays an important role across several fields, including synthetic org. chem., materials science, targeted drug delivery and the probing of biol. function. Although a myriad of methods now exist for the modification of peptides or proteins, many of these target a handful of the most reactive proteinogenic amino acids. Photocatalysis has recently emerged as a mild approach for amino acid modification, generating a sizable toolbox of reactions capable of modifying almost all of the canonical amino acids. These reactions are characterised by their mild, physiol. compatible conditions, greatly enhancing their usefulness for amino acid modification. This review aims to introduce the field of photocatalytic amino acid modification and discusses the most recent advances.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit12ktbnL&md5=8d310120eb6697790104b242e51b0609
      Bloom, S.; Liu, C.; Kolmel, D. K.; Qiao, J. X.; Zhang, Y.; Poss, M. A.; Ewing, W. R.; MacMillan, D. W. C. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 2018, 10 (2), 205– 211,  DOI: 10.1038/nchem.2888
      14
      Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials
      Bloom, Steven; Liu, Chun; Kolmel, Dominik K.; Qiao, Jennifer X.; Zhang, Yong; Poss, Michael A.; Ewing, William R.; MacMillan, David W. C.
      Nature Chemistry (2018), 10 (2), 205-211CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      The advent of antibody-drug conjugates as pharmaceuticals has fueled 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 soln. to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, the authors explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, the authors demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidn. potentials between internal vs. C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidn. potential-gated technol. is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodol. provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWgsrjJ&md5=a5f8b7f701329a625221c77280243746
    15. 15
      Vohidov, F.; Coughlin, J. M.; Ball, Z. T. Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains. Angew. Chem., Int. Ed. Engl. 2015, 54 (15), 4587– 4591,  DOI: 10.1002/anie.201411745
      There is no corresponding record for this reference.
      Peciak, K.; Laurine, E.; Tommasi, R.; Choi, J. W.; Brocchini, S. Site-selective protein conjugation at histidine. Chemical Science 2019, 10 (2), 427– 439,  DOI: 10.1039/C8SC03355B
      There is no corresponding record for this reference.
      Nakane, K.; Sato, S.; Niwa, T.; Tsushima, M.; Tomoshige, S.; Taguchi, H.; Ishikawa, M.; Nakamura, H. Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen. J. Am. Chem. Soc. 2021, 143 (20), 7726– 7731,  DOI: 10.1021/jacs.1c01626
      15
      Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen
      Nakane, Keita; Sato, Shinichi; Niwa, Tatsuya; Tsushima, Michihiko; Tomoshige, Shusuke; Taguchi, Hideki; Ishikawa, Minoru; Nakamura, Hiroyuki
      Journal of the American Chemical Society (2021), 143 (20), 7726-7731CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small mol., 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liq. chromatog.-mass spectrometry anal. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpsFehs7Y%253D&md5=d9bac378e73ba9c624abd20cb6489a23
      Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi, I. One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity. J. Am. Chem. Soc. 2006, 128 (10), 3273– 3280,  DOI: 10.1021/ja057926x
      15
      One-Pot and Sequential Organic Chemistry on an Enzyme Surface to Tether a Fluorescent Probe at the Proximity of the Active Site with Restoring Enzyme Activity
      Takaoka, Yousuke; Tsutsumi, Hiroshi; Kasagi, Noriyuki; Nakata, Eiji; Hamachi, Itaru
      Journal of the American Chemical Society (2006), 128 (10), 3273-3280CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A new and simple method to tether a functional mol. at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivs., so that it restored the enzymic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping expt. revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chem., the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1CitbY%253D&md5=959d5fc717b79240bad70c3d7e12feef
    16. 16
      Labroo, V. M.; Labroo, R. B.; Cohen, L. A. Direct Photochemical Trifluoromethylation of Histidine-Containing Peptides. Tetrahedron Lett. 1990, 31 (40), 5705– 5708,  DOI: 10.1016/S0040-4039(00)97937-1
      16
      Direct photochemical trifluoromethylation of histidine-containing peptides
      Labroo, V. M.; Labroo, R. B.; Cohen, L. A.
      Tetrahedron Letters (1990), 31 (40), 5705-8CODEN: TELEAY; ISSN:0040-4039.
      Photochem. trifluoromethylation of the imidazole ring of histidine in the tripeptide Glp-His-Pro-NH2 has been achieved to furnish a mixt. of imidazole trifluoromethylated isomers, which have been sepd. by reverse-phase HPLC and characterized.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXisVGruw%253D%253D&md5=972dbf43e25be716d0bddbe5d1f1f930
      Wan, C.; Wang, Y.; Lian, C.; Chang, Q.; An, Y.; Chen, J.; Sun, J.; Hou, Z.; Yang, D.; Guo, X. Histidine-specific bioconjugation via visible-light-promoted thioacetal activation. Chem. Sci. 2022, 13 (28), 8289– 8296,  DOI: 10.1039/D2SC02353A
      16
      Histidine-specific bioconjugation via visible-light-promoted thioacetal activation
      Wan, Chuan; Wang, Yuena; Lian, Chenshan; Chang, Qi; An, Yuhao; Chen, Jiean; Sun, Jinming; Hou, Zhanfeng; Yang, Dongyan; Guo, Xiaochun; Yin, Feng; Wang, Rui; Li, Zigang
      Chemical Science (2022), 13 (28), 8289-8296CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addn. to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine contg. proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhs12ls7jI&md5=30b9b2abd14a803c14a2c241ccf04b95
      Chen, X.; Ye, F.; Luo, X.; Liu, X.; Zhao, J.; Wang, S.; Zhou, Q.; Chen, G.; Wang, P. Histidine-Specific Peptide Modification via Visible-Light-Promoted C-H Alkylation. J. Am. Chem. Soc. 2019, 141 (45), 18230– 18237,  DOI: 10.1021/jacs.9b09127
      16
      Histidine-specific peptide modification via visible-light-promoted C-H alkylation
      Chen, Xiaoping; Ye, Farong; Luo, Xiaosheng; Liu, Xueyi; Zhao, Jie; Wang, Siyao; Zhou, Qingqing; Chen, Gong; Wang, Ping
      Journal of the American Chemical Society (2019), 141 (45), 18230-18237CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Histidine (His) carries a unique heteroarom. imidazole side chain and plays irreplaceable functional roles in peptides and proteins. Existing strategies for site-selective histidine modification predominantly rely on the N-substitution reactions of the moderately nucleophilic imidazole group, which inherently suffers from the interferences from lysine and cysteine residues. Chemoselective modification of histidine remains one of the most difficult challenges in peptide chem. Herein, we report peptide modification via radical-mediated chemoselective C-H alkylation of histidine using C4-alkyl-1,4-dihydropyridine (DHP) reagents under visible-light-promoted conditions. The method exploits the electrophilic reactivity of the imidazole ring via a Minisci-type reaction pathway. This method exhibits an exceptionally broad scope for both peptides and DHP alkylation reagents. Its utility has been demonstrated in a series of important peptide drugs, complex natural products, and a small protein. Distinct from N-substitution reactions, the unsubstituted nitrogen groups of the modified imidazole ring are conserved in the C-H alkylated products.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvF2kurvE&md5=3625d153fe10e6ad4fde7786097f9dd0
      Jia, S.; He, D.; Chang, C. J. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. J. Am. Chem. Soc. 2019, 141 (18), 7294– 7301,  DOI: 10.1021/jacs.8b11912
      16
      Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation
      Jia, Shang; He, Dan; Chang, Christopher J.
      Journal of the American Chemical Society (2019), 141 (18), 7294-7301CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. The authors now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic posttranslational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddn. (CuAAC) chem. The authors establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purifn., as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chem.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnvFCgsL8%253D&md5=57725240e216d1c915749ed964081b13
      Uchida, K.; Stadtman, E. R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544– 4548,  DOI: 10.1073/pnas.89.10.4544
      16
      Modification of histidine residues in proteins by reaction with 4-hydroxynonenal
      Uchida, Koji; Stadtman, E. R.
      Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (10), 4544-8CODEN: PNASA6; ISSN:0027-8424.
      Histidine residues in proteins are major targets for reaction with the lipid peroxidn. product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivs. upon subsequent treatment with NaB[3H]H4. Amino acid anal. of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stoichiometric formation of 3H-labeled amino acid hydrolyzates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the prodn. of two compds. Upon acid hydrolysis, both derivs. yielded stoichiometric amts. of histidine. However, after redn. with NaBH4, acid hydrolysis led to a mixt. of amino acid derivs. [presumably, isomeric forms of Nπ(Nτ)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addn. of the imidazole nitrogen atom of histidine to the α,β-unsatd. bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be detd.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVeqtL4%253D&md5=e319c0a1200aa40784b00fde9bdd7cd6
    17. 17
      Joshi, P. N.; Rai, V. Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification. Chem. Commun. (Camb) 2019, 55 (8), 1100– 1103,  DOI: 10.1039/C8CC08733D
      There is no corresponding record for this reference.
      Rawale, D. G.; Thakur, K.; Sreekumar, P.; Sajeev, T. K.; Ramesh, A.; Adusumalli, S. R.; Mishra, R. K.; Rai, V. Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins. Chem. Sci. 2021, 12 (19), 6732– 6736,  DOI: 10.1039/D1SC00335F
      17
      Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins
      Rawale, Dattatraya Gautam; Thakur, Kalyani; Sreekumar, Pranav; T. K., Sajeev; A., Ramesh; Adusumalli, Srinivasa Rao; Mishra, Ram Kumar; Rai, Vishal
      Chemical Science (2021), 12 (19), 6732-6736CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create a unique chem. space to enable site-selectivity for histidine and aspartic acid modifications overcoming the pre-requisite of chemoselectivity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotlygs7w%253D&md5=a13bce7fd4e21e1865e6b7823ecf164c
    18. 18
      Drienovska, I.; Rioz-Martinez, A.; Draksharapu, A.; Roelfes, G. Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids. Chem. Sci. 2015, 6 (1), 770– 776,  DOI: 10.1039/C4SC01525H
      There is no corresponding record for this reference.
    19. 19
      Huard, D. J.; Kane, K. M.; Tezcan, F. A. Re-engineering protein interfaces yields copper-inducible ferritin cage assembly. Nat. Chem. Biol. 2013, 9 (3), 169,  DOI: 10.1038/nchembio.1163
      19
      Re-engineering protein interfaces yields copper-inducible ferritin cage assembly
      Huard, Dustin J. E.; Kane, Kathleen M.; Tezcan, F. Akif
      Nature Chemical Biology (2013), 9 (3), 169-176CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      The ability to chem. control protein-protein interactions would allow the interrogation of dynamic cellular processes and lead to a better understanding and exploitation of self-assembling protein architectures. Here we introduce a new engineering strategy - reverse metal-templated interface redesign (rMeTIR) - that transforms a natural protein-protein interface into one that only engages in selective response to a metal ion. We have applied rMeTIR to render the self-assembly of the cage-like protein ferritin controllable by divalent copper binding, which has allowed the study of the structure and stability of the isolated ferritin monomer, the demonstration of the primary role of conserved hydrogen-bonding interactions in providing geometric specificity for cage assembly and the uniform chem. modification of the cage interior under physiol. conditions. Notably, copper acts as a structural template for ferritin assembly in a manner that is highly reminiscent of RNA sequences that template virus capsid formation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFKqsL8%253D&md5=e52b99e1124df8ac2892b12e70f2e655
    20. 20
      Hempstead, P. D.; Yewdall, S. J.; Fernie, A. R.; Lawson, D. M.; Artymiuk, P. J.; Rice, D. W.; Ford, G. C.; Harrison, P. M. Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 1997, 268 (2), 424– 448,  DOI: 10.1006/jmbi.1997.0970
      20
      Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution
      Hempstead, Paul D.; Yewdall, Stephen J.; Fernie, Alisdair R.; Lawson, David M.; Artymiuk, Peter J.; Rice, David W.; Ford, Geoffrey C.; Harrison, Pauline M.
      Journal of Molecular Biology (1997), 268 (2), 424-448CODEN: JMOBAK; ISSN:0022-2836. (Academic)
      Mammalian ferritins are 24-mers assembled from two types of polypeptide chain which provide the mol. with different functions. Heavy (H) chains catalyze the first step in iron storage, the oxidn. of iron(II). Light (L) chains promote the nucleation of the mineral ferrihydrite enabling storage of iron(III) inside the protein shell. We report here the comparison of the three-dimensional structures of recombinant human H chain (HuHF) and horse L chain (HoLF) ferritin homopolymers, which have been refined at 1.9 Å resoln. There is 53% sequence identity between these mols., and the two structures are very similar, the H and L subunit α-carbons superposing to within 0.5 Å rms deviation with 41 water mols. in common. Nevertheless, there are significant important differences which can be related to differences in function. In particular, the centers of the four-helix bundles contain distinctive groups of hydrophilic residues which have been assocd. with ferroxidase activity in H chains and enhanced stability in L chains. L chains contain a group of glutamates assocd. with mineralization within the iron storage cavity of the protein.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjsValuro%253D&md5=ad1c56bf687a2c402329f517c58c2eed
    21. 21
      Xiao, H.; Peters, F. B.; Yang, P. Y.; Reed, S.; Chittuluru, J. R.; Schultz, P. G. Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase. ACS Chem. Biol. 2014, 9 (5), 1092– 1096,  DOI: 10.1021/cb500032c
      21
      Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase
      Xiao, Han; Peters, Francis B.; Yang, Peng-Yu; Reed, Sean; Chittuluru, Johnathan R.; Schultz, Peter G.
      ACS Chemical Biology (2014), 9 (5), 1092-1096CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogs in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase (PylRS) mutant was able to site-specifically incorporate 3-methylhistidine, 3-pyridylalanine, 2-furylalanine, and 3-(2-thienyl)alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogs created mutant proteins with distinct spectral properties. This work further expands the structural and chem. diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVOqt70%253D&md5=d94387a5111558bb29264e40f2572570
    22. 22
      Wang, Y.-S.; Fang, X.; Wallace, A. L.; Wu, B.; Liu, W. R. A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. J. Am. Chem. Soc. 2012, 134 (6), 2950– 2953,  DOI: 10.1021/ja211972x
      22
      A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a Broad Substrate Spectrum
      Wang, Yane-Shih; Fang, Xinqiang; Wallace, Ashley L.; Wu, Bo; Liu, Wenshe R.
      Journal of the American Chemical Society (2012), 134 (6), 2950-2953CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Together with tRNACUAPyl, a rationally designed pyrrolysyl-tRNA synthetase (PylRS) mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivs. with large para substituents into superfolder green fluorescent protein (sfGFP) at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivs. using engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pairs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVyktLg%253D&md5=d5f9189997497a11a60b743c0020dda3
    23. 23
      Zaykov, A. N.; Mayer, J. P.; DiMarchi, R. D. Pursuit of a perfect insulin. Nat. Rev. Drug Discov 2016, 15 (6), 425– 439,  DOI: 10.1038/nrd.2015.36
      There is no corresponding record for this reference.
    24. 24
      Menting, J. G.; Whittaker, J.; Margetts, M. B.; Whittaker, L. J.; Kong, G. K. W.; Smith, B. J.; Watson, C. J.; Záková, L.; Kletvíková, E.; Jirácek, J. How insulin engages its primary binding site on the insulin receptor. Nature 2013, 493 (7431), 241– U276,  DOI: 10.1038/nature11781
      24
      How insulin engages its primary binding site on the insulin receptor
      Menting, John G.; Whittaker, Jonathan; Margetts, Mai B.; Whittaker, Linda J.; Kong, Geoffrey K.-W.; Smith, Brian J.; Watson, Christopher J.; Zakova, Lenka; Kletvikova, Emilia; Jiracek, Jiri; Chan, Shu Jin; Steiner, Donald F.; Dodson, Guy G.; Brzozowski, Andrzej M.; Weiss, Michael A.; Ward, Colin W.; Lawrence, Michael C.
      Nature (London, United Kingdom) (2013), 493 (7431), 241-245CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Insulin receptor signaling has a central role in mammalian biol., regulating cellular metab., growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signaling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titrn. calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochem. data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXls1yquw%253D%253D&md5=4e4b1abc994ed5944becebe53034d840

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c14446.

    • List of abbreviations, Ftn variants, peptide/protein sequences, plasmids and primers, Materials and Method, Supplementary Tables S1–S4, and Supplementary Figures S1–S35, including results from MS, X-ray crystallography, Cryo-EM, PAGE, DLS and TEM analyses (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.