Pruning the ALS-Associated Protein SOD1 for in-Cell NMR

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• 作者：Jens Danielsson ; Kohsuke Inomata ; Shuhei Murayama ; Hidehito Tochio ; Lisa Lang ; Masahiro Shirakawa ; Mikael Oliveberg
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：July 17, 2013
• 年：2013
• 卷：135
• 期：28
• 页码：10266-10269
• 全文大小：263K
• 年卷期：v.135,no.28(July 17, 2013)
• ISSN：1520-5126

文摘

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^螖IV螖VII) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1^螖IV螖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^螖IV螖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 尾 strands 4, 5, and 7, triggered by partial protonation of the His moieties of the Cu-binding site. Accordingly, SOD1^螖IV螖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.