Year-end Sale % OFF 
Abstract
The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. For some proteins, the stability increases, while for others, it decreases, depending on both the sequence composition and the type of host cell. The mechanism seems to be linked to the strength and conformational bias of the diffusive in-cell interactions, where protein charge is found to play a decisive role. Because most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment behaves like a polyanionic (Z:1) system with electrostatic interactions different from those of standard 1:1 ion solutes. To determine how such polyanion conditions influence protein stability, we use negatively charged polyacetate ions to mimic the net-negatively charged cellular environment. The results show that, per Na+ equivalent, polyacetate destabilizes the model protein SOD1barrel significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na+, the polyacetate destabilization of SOD1barrel is similar to that observed in live cells. By the combined use of equilibrium thermal denaturation, folding kinetics, and high-resolution nuclear magnetic resonance, this destabilization is primarily assigned to preferential interaction between polyacetate and the globally unfolded protein. This interaction is relatively weak and involves mainly the outermost N-terminal region of unfolded SOD1barrel. Our findings point thus to a generic influence of polyanions on protein stability, which adds to the sequence-specific contributions and needs to be considered in the evaluation of in vivo data.
Highlights
The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells
The focus of this study is to examine how polyanions modulate protein stability by asking the simplest question conceivable: what is the effect of linking a given number of solute 1:1 ions into Z:1 ions? As a model protein, we chose the well-characterized SOD1barrel (Figure S1)[3,6,16,32−34] with the destabilizing core mutation I35A (SOD1I35A).[6]
I35A mutation is to move the thermal unfolding transition into the physiological regime, which allows for a direct characterization of the curved ΔG°U−F versus T profile (Figure 2), as well as a direct comparison with previously published in-cell nuclear magnetic resonance (NMR) data.[6]
Summary
The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. Subsequent transfer of SOD1barrel from human cells to the interior of Escherichia coli decreases ΔH°U−F(Tm) and the maximum stability even further, but with an accompanying increase in Tm (Figure 1).[6] Such a contrasting thermodynamic signature suggests that, on top of the destabilization caused by preferential binding, there is a stabilizing term from excluded-volume effects.[17] Given that the cytosol of E. coli cells is 3−6 times more crowded than that of human cells, this result is perfectly reasonable.[18,5] The data suggest, interestingly, that the same molecular determinants are at play, both in the native eukaryotic environment and in the E. coli cytosol, in various relative amounts. This repulsive situation will basically oppose association and favor protein dispersion.[1,14]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
