Understanding Supramolecular Assembly of Supercharged Proteins

Abstract

Ordered supramolecular assemblies have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly of oppositely supercharged proteins are not fully understood. Here, 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 variants. We show that 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), but the assembled supramolecular structures critically depend on surface charge distributions. Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues 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 further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of ∼10–40 min, which is governed by protein composition and solution conditions. Broadly, our results inform how protein supercharging can be used to create different ordered supramolecular assemblies from a single parent protein building block.

Supercharged Fluorescent Protein-Apoferritin Cocrystals for Lighting Applications

“…As a control, the same experimental conditions were applied to prepare cocrystals of mGL and aFt without any success (Figure S6), as expected by the negative surface net charge of mGL (i.e., −2). Thus, our scmGL design, in which the majority of the positive charges are located at the protein loops (Figure B), enables strong interaction through long-range electrostatic interactions driving the formation of the assembly …”

Assembly Requirements for the Construction of Large-Scale Binary Protein Structures

“…For the assembly of supercharged green fluorescent proteins (GFP) into discrete structures, a follow-up study investigated the role of the introduced mutations . Similar to our findings, both interface-mediating residues and long-range interactions are needed to form discrete GFP assemblies.…”

“…For the assembly of supercharged green fluorescent proteins (GFP) into discrete structures, a follow-up study investigated the role of the introduced mutations. 70 Similar to our findings, both interface-mediating residues and long-range interactions are needed to form discrete GFP assemblies. However, for the ferritin system, we performed a full screening of all variants, including combinations of proteins containing just interfacemediating residues or only one charged patch in total.…”

Supercharged Fluorescent Protein-Apoferritin Cocrystals for Lighting Applications

“…Thus, our scmGL design, in which the majority of the positive charges are located at the protein loops ( Figure 2B ), enables strong interaction through long-range electrostatic interactions driving the formation of the assembly. 41…”

“…Finally, we consider those mutations that are highly exposed to reduce interprotein interaction that might reduce long-range electrostatic interaction in the cocrystallization process. 41 All-in-all, our strategy leads us to design eleven mutations: eight on the flexible protein loops and three on two β-sheets (Figure 2A-B). This results in a theoretical protein charge of +22 without considering the terminal histidine tag (i.e., +28 in total).…”