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bioRxiv · 10.1101/2022.06.21.497010

Understanding Supramolecular Assembly of Supercharged Proteins

Jacobs, M. I.·Bansal, P.·Shukla, D.·Schroeder, C. M.
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Abstract

Ordered supramolecular assemblies of supercharged synthetic proteins have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly process between oppositely supercharged proteins are not fully understood. In this work, we use a combination of experiments and computational modeling to systematically study the supramolecular assembly process for a series of oppositely supercharged green fluorescent protein (GFP) variants. Our results show that the assembled structures of oppositely supercharged proteins critically depend on surface charge distributions. In addition, net charge is a sufficient molecular descriptor to predict the interaction fate of oppositely charged proteins under a given set of solution conditions (e.g., ionic strength). Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues that are not directly involved in interprotein interactions contribute to a substantial fraction ([~]30%) of the interaction energy between oppositely charged proteins via long-range electrostatic interactions. Dynamic subunit exchange experiments enabled by Forster resonance energy transfer (FRET) further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of [~]10-40 minutes, which is governed by protein composition and solution conditions. Overall, our work shows that a balance between kinetic stability and electrostatic charge ultimately determine the fate of supramolecular assemblies of supercharged proteins. Broadly, our results inform how protein supercharging can be used to generate different ordered supramolecular assemblies from a single parent protein building block.

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Jacobs, M. I., Bansal, P., Shukla, D., Schroeder, C. M.. 2022-06-21. Understanding Supramolecular Assembly of Supercharged Proteins. https://doi.org/10.1101/2022.06.21.497010

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