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Pruning the ALS-Associated Protein SOD1 for in-Cell NMR
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2013 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 28, 10266-10269 p.Article in journal (Refereed) Published
Abstract [en]

To efficiently deliver isotope-labeled 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 deliver the ALS-associated protein superoxide dismutase (SOD1) into HeLa cells. Our results show that, although full-length SOD1 cannot be efficiently internalized, a variant in which the active-site loops IV and VII have been truncated (SOD1(Delta IV Delta VII))) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1(Delta IV Delta 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 internalized SOD1(Delta IV Delta VII) protein displays high-resolution in-cell NMR spectra similar to, but not identical to, those of the lysate of the cells. Spectral differences are found mainly in the dynamic beta strands 4, 5, and 7, triggered by partial protonation of the His moieties of the Cu-binding site. Accordingly, SOD1(Delta IV Delta VII) doubles here as an internal pH probe, revealing cytosolic acidification under the experimental treatment. Taken together, these observations show that CPP delivery, albeit inefficient at first trials, can be tuned by protein engineering to allow atomic-resolution NMR studies of specific protein structures that have evaded other in-cell NMR approaches: in this case, the structurally elusive apoSOD1 barrel implicated as precursor for misfolding in ALS.

Place, publisher, year, edition, pages
2013. Vol. 135, no 28, 10266-10269 p.
National Category
Biological Sciences
URN: urn:nbn:se:su:diva-94051DOI: 10.1021/ja404425rISI: 000322103000022OAI: diva2:651072


Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2013-09-24Bibliographically approved

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Danielsson, JensLang, LisaOliveberg, Mikael
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Department of Biochemistry and Biophysics
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