Atomic-resolution monitoring of protein maturation in live human cells by NMR

Abstract

We used NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We could follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 (CCS) oxidation of the SOD1 intrasubunit disulfide bond occurs through both copper-dependent and independent mechanisms. Our approach represents a new strategy for structural investigation of endogeneously expressed proteins within a physiological (cellular) environment.

Protein dynamics and function from solution state NMR spectroscopy

“…inside living cells. 'In-cell' and 'in-lysate' NMR spectroscopy (Danielsson et al 2013(Danielsson et al , 2015Serber et al 2005Serber et al , 2007Smith et al 2013) have developed to a point where new aspects of protein function can be revealed (Banci et al 2013). For instance in a recent study Selenko and co-workers developed an NMR-based methodology that enables quantification of the temporal phosphorylation and de-phosphorylation of short protein segments in cell lysates (Thongwichian et al 2015).…”

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

“…3), as observed for WT SOD1 (ref. 10). Chemical shift analysis shows that for the latter mutants only minor changes occur in the twodimensional (2D) NMR spectra, indicating that the overall structure is maintained in the E,Zn-SOD1 form, as the mutations only cause small local perturbations on the protein conformation ( Supplementary Fig.…”

“…10). With this approach, proteins are overexpressed and isotopically labelled in cultured human cells, and information on the intracellular folding, cysteine oxidation and metallation state of the protein is obtained by observing them directly in living cells by NMR.…”

“…This level of characterization is necessary to understand the molecular cause of cell malfunction in pathological states and to develop treatments and therapeutic protocols. We have recently developed an in-cell NMR approach aimed at obtaining information directly in live human cells on the folding and maturation process of proteins as they are produced 10,11 . This in-human-cell NMR approach was applied to monitor maturation events for human proteins such as Mia40 (ref.…”

“…This in-human-cell NMR approach was applied to monitor maturation events for human proteins such as Mia40 (ref. 11) and superoxide dismutase 1 (SOD1) 10 . The latter is a stable dimer binding a catalytic copper ion and a structural zinc ion per monomer, and forming an intramolecular disulfide bond.…”