A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly

Ahmad, Raheel; Tyryshkin, Alexei M.; Xie, Lingjun; Hansen, William A.; Yachnin, Brahm J.; Emge, Thomas J.; Mashrai, Ashraf; Khare, Sagar D.; Knapp, Spencer

doi:10.1016/j.jinorgbio.2023.112206

A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly

Journal Article · Sat Apr 01 00:00:00 EDT 2023 · Journal of Inorganic Biochemistry

DOI:https://doi.org/10.1016/j.jinorgbio.2023.112206· OSTI ID:2423260

Ahmad, Raheel ^[1]; Tyryshkin, Alexei M. ^[2]; Xie, Lingjun ^[1]; Hansen, William A. ^[3]; Yachnin, Brahm J. ^[3]; Emge, Thomas J. ^[1]; Mashrai, Ashraf ^[3]; Khare, Sagar D. ^[3]; Knapp, Spencer ^[1]

Rutgers Univ., Piscataway, NJ (United States)
Rutgers Univ., New Brunswick, NJ (United States)
Rutgers Univ., Piscataway, NJ (United States); Rutgers Center for Integrative Proteomics Research, Piscataway, NJ (United States)

Precise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated. Here, we expand the biomolecular metal-coordination toolbox with the irreversible attachment to cysteine of bis(1-methylimidazol-2-yl)ethene (“BMIE”), which generates a compact imidazole-based metal-coordinating ligand. Conjugate additions of small-molecule thiols (thiocresol and N-Boc-Cys) with BMIE confirm general thiol reactivity. The BMIE adducts are shown to complex the divalent metal ions Cu⁺⁺ and Zn⁺⁺ in bidentate (N₂) and tridentate (N₂S*) coordination geometries. Cysteine-targeted BMIE modification (>90% yield at pH 8.0) of a model protein, the S203C variant of carboxypeptidase G2 (CPG2), measured with ESI-MS, confirms its utility as a site-selective bioconjugation method. ICP-MS analysis confirms mono-metallation of the BMIE-modified CPG2 protein with Zn⁺⁺, Cu⁺⁺, and Co⁺⁺. EPR characterization of the BMIE-modified CPG2 protein reveals the structural details of the site selective 1:1 BMIE-Cu⁺⁺ coordination and symmetric tetragonal geometry under physiological conditions and in the presence of various competing and exchangeable ligands (H₂O/HO^–, tris, and phenanthroline). An X-ray protein crystal structure of BMIE-modified CPG2-S203C demonstrates that the BMIE modification is minimally disruptive to the overall protein structure, including the carboxypeptidase active sites, although Zn⁺⁺ metalation could not be conclusively discerned at the resolution obtained. The carboxypeptidase catalytic activity of BMIE-modified CPG2-S203C was also assayed and found to be minimally affected. Finally, these features, combined with ease of attachment, define the new BMIE-based ligation as a versatile metalloprotein design tool, and enable future catalytic and structural applications.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2423260

Alternate ID(s):: OSTI ID: 1968642

Journal Information:: Journal of Inorganic Biochemistry, Journal Name: Journal of Inorganic Biochemistry Vol. 244; ISSN 0162-0134

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly

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