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

Abstract

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(ΔIVΔVII)) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1(ΔIVΔVII) 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(ΔIVΔVII) 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 β strands 4, 5, and 7, triggered by partial protonation of the His moieties of the Cu-binding site. Accordingly, SOD1(ΔIVΔVII) 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.

Equilibrium simulation of trp-cage in the presence of protein crowders

“…These studies have, in particular, shown that crowding can cause an either upward or downward shift in the stability of folded test proteins, [7][8][9][11][12][13][14][15][16] and the direction of the shift can, in part, be rationalized based on net-charge considerations. 9 The protein of the present study, trp-cage, has a well-defined folded structure but is small and malleable.…”

“…6 Today, experiments are increasingly often performed under more realistic crowding conditions, using concentrated protein solutions [7][8][9][10] or cells. 9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins. [7][8][9][12][13][14]16 For a fundamental understanding of these effects, there is a need for computational approaches that permit the simulation of folding/unfolding equilibria in the presence of complex biomolecular crowding agents.…”

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

“…I n-cell NMR represents a unique tool for characterizing physiological processes and their alteration in the 'natural' environment, that is, within a live cellular context, with an atomic-level approach [1][2][3][4][5][6][7][8][9] . This level of characterization is necessary to understand the molecular cause of cell malfunction in pathological states and to develop treatments and therapeutic protocols.…”