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In Vitro N-Terminal Acetylation of Bacterially Expressed Parvalbumins by N-Terminal Acetyltransferases from Escherichia coli

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Abstract

Most eukaryotic proteins are N-terminally acetylated (Nt-acetylated) by specific N-terminal acetyltransferases (NATs). Although this co-/post-translational protein modification may affect different aspects of protein functioning, it is typically neglected in studies of bacterially expressed eukaryotic proteins, lacking this modification. To overcome this limitation of bacterial expression, we have probed the efficiency of recombinant Escherichia coli NATs (RimI, RimJ, and RimL) with regard to in vitro Nt-acetylation of several parvalbumins (PAs) expressed in E. coli. PA is a calcium-binding protein of vertebrates, which is sensitive to Nt-acetylation. Our analyses revealed that only metal-free PAs were prone to Nt-acetylation (up to 100%), whereas Ca2+ binding abolished this modification, thereby indicating that Ca2+-induced structural stabilization of PAs impedes their Nt-acetylation. RimJ and RimL were active towards all PAs with N-terminal serine. Their activity towards PAs beginning with alanine was PA-specific, suggesting the importance of the subsequent residues. RimI showed the least activity regardless of the PA studied. Overall, NATs from E. coli are suited for post-translational Nt-acetylation of bacterially expressed eukaryotic proteins with decreased structural stability.

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Acknowledgments

We are grateful to Rohan T. Baker for providing us with the pHUE and pHUsp2-cc plasmids.

Funding

The work was supported by a grant to Y.S.L. from the Russian foundation for basic research (No. 18-34-00701).

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Authors and Affiliations

  1. Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, Pushchino, Moscow Region, 142290, Russia

    Yulia S. Lapteva, Alisa A. Vologzhannikova, Andrey S. Sokolov, Ramis G. Ismailov, Vladimir N. Uversky & Sergei E. Permyakov

  2. Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA

    Vladimir N. Uversky

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  1. Yulia S. Lapteva
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  2. Alisa A. Vologzhannikova
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Lapteva, Y.S., Vologzhannikova, A.A., Sokolov, A.S. et al. In Vitro N-Terminal Acetylation of Bacterially Expressed Parvalbumins by N-Terminal Acetyltransferases from Escherichia coli. Appl Biochem Biotechnol 193, 1365–1378 (2021). https://doi.org/10.1007/s12010-020-03324-8

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