[NMR paper] Pruning the ALS-associated protein SOD1 for in-cell NMR.

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Pruning the ALS-associated protein SOD1 for in-cell NMR.

Related Articles Pruning the ALS-associated protein SOD1 for in-cell NMR.

J Am Chem Soc. 2013 Jul 2;

Authors: Danielsson J, Inomata K, Murayama S, Tochio H, Lang L, Shirakawa M, Oliveberg M

Abstract
To efficiently deliver isotope-labelled proteins into mammalian cells poses a main challenge for structural and functional analysis by in-cell NMR. In this study we have employed cell-penetrating peptides (CPPs) to transfer the ALS-associated protein superoxide dismutase (SOD1) into HeLa cells. Our results show that, although full-length SOD1 cannot be efficiently internalised, a variant in which the active-site loops IV and VII have been truncated (SOD1?IV ?VII) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1?IV ?VII seems to be the elimination of negatively charged side chains, which alters the net charge of the CPP-SOD1 complex from neutral to +4. The internalised SOD1?IV ?VII protein displays high-resolution in-cell NMR spectra similar to, but not identical to, those of the folded state outside cells. Spectral differences are found mainly in the dynamic ? strands 4, 5 and 7, triggered by partial protonation of the His moieties of the Cu-binding site. As such SOD1?IV ?VII doubles here as an internal pH probe, revealing cytosolic acidification under the experimental treatment. Taken together, these observations show that CPP delivery, albeit at first trials inefficient, can be tuned by protein engineering to allow atomic-resolution NMR studies of specific protein structures that has evaded other in-cell NMR approaches: in this case the structurally elusive apoSOD1 barrel implicated as precursor for misfolding in ALS.


PMID: 23819500 [PubMed - as supplied by publisher]



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