Abstract
In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes.
Highlights
Folding and maturation of proteins characterized by posttranslational modifications and formation of quaternary structure is a complex process which progresses through a number of well concerted events
It is physiologically expressed at relatively high concentrations in human cells, and it exerts its function in the cytoplasm, in the nucleus and in the mitochondrial IMS [7]
The process leading a newly synthesized protein to acquire its final, functional state could involve several steps which consist of protein folding to its tertiary and possibly quaternary structure, cofactor binding and post-translational modifications
Summary
Folding and maturation of proteins characterized by posttranslational modifications and formation of quaternary structure is a complex process which progresses through a number of well concerted events. It has been previously shown that the bacterial cytoplasm is a good model of the eukaryotic one, especially to study the effects of molecular crowding on protein folding and non-specific interactions [3], as they have similar pH and redox potential [4,5,6]. Within this frame, we have characterized by in-cell NMR the wild-type human copper, zinc superoxide dismutase 1 (hSOD1) protein, as well as the initial steps towards its maturation. Two conserved cysteine residues (Cys 57 and 146) form an intramolecular disulfide bridge during the protein maturation process
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