Abstract
We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.
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Acknowledgements
We would like to thank E.Y. Jones and D.I. Stuart for critically reading the manuscript and providing advice. This work was supported by the Access to Research Infrastructures activities in the Seventh Framework Programme of the European Commission (Bio-NMR contract 261863 and P-CUBE contract 227764), by the Italian grant program MIUR-PRIN 2009 'Biologia strutturale meccanicistica: avanzamenti metodologici e biologici' and by the European Integrated Structural Biology Infrastructure (INSTRUCT), which is part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions. Specifically, we thank the INSTRUCT Core Centres CERM (Italy) and the University of Oxford. A.R.A. was supported by a UK Medical Research Council Career Development Award fellowship.
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L. Banci, I.B. and A.R.A. conceived the work; L. Banci, L. Barbieri, E.L. and A.R.A. designed the experiments; L. Barbieri and Y.Z. grew and transfected the human cells; L. Barbieri cloned the genes and produced the cell samples of SOD1, CCS, Atox1, Grx1 and Trx; E.S. produced the cell sample of Mia40; L. Barbieri and E.L. produced and analyzed the E. coli cell samples; E.L. performed the NMR experiments and analyzed the data; L. Banci, L. Barbieri, I.B., E.L. and A.R.A. wrote the paper.
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Banci, L., Barbieri, L., Bertini, I. et al. Atomic-resolution monitoring of protein maturation in live human cells by NMR. Nat Chem Biol 9, 297–299 (2013). https://doi.org/10.1038/nchembio.1202
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DOI: https://doi.org/10.1038/nchembio.1202
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