Supercharging enables organized assembly of synthetic biomolecules

Abstract

Abstract:There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16-mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/323261 doi: bioRxiv preprint first posted online May. 16, 2018; 2 The assembly of genetically-encoded molecules into symmetric, supramolecular materials enables complex functions in natural biosystems and would likewise prove valuable in the development of bionanotechnologies including drug delivery, energy transport, and biological information storage. [1][2][3][4][5][6][7] Repeated, symmetric interactions between molecular building blocks enable the formation of complex, defined, assemblies from a small total number of components as well as dynamic propagation of conformational change. [3][4][5][6][7][8] However, while de novo engineering of artificial symmetrical biomolecular complexes has begun to be explored, 9-15 these efforts generally rely upon precise design of molecular interfaces. 16,17 In contrast, studies of simple synthetic colloids suggests that higher order structures can be derived based solely on packing and energetic and considerations. Computational and experimental studies of polyhedral or spherical colloids indicate that complementary particle shapes 18-24 and simple attractive "patches" [24][25][26][27][28][29] alone can enable formation of complex symmetrical assemblies. Shape complementarity has likewise been exploited to arrange synthetic linear biomolecules, i.e., double-stranded DNA strands, into distinct structures, demonstrating its utility for for the higher order arrangement of synthetic linear biomolecules, suggesting a utility beyond inorganic systems. 30,31Herein we demonstrate a simple, robust strategy to assemble normally monomeric proteins into well defined, oligomeric quaternary structures and micron-scale particles. This strategy centers on driving protein interactions by engineering oppositely supercharged variants. Generally, oppositely-charged proteins interact through simple electrostativ interactions. 32 Previously, this propensity has been exploited in artificial biosystems to engineer binary protein crystals from naturally oppos...

Thermodynamically driven assemblies and liquid–liquid phase separations in biology

“…In such representations, individual proteins are treated as colloidal particles interacting via isotropic [228] or patchy [229,230] potentials. The interest in probing the thermodynamics and kinetics of phase transitions in patchy colloidal systems [231][232][233][234][235][236], however, extends far beyond understanding protein crystallization, as patchy colloids are excellent model systems for inspecting how a competition between isotropic and directional interactions can impact the phase behavior, e.g., the relative stability of different crystals and liquids, as well as the kinetics of self-assembly.…”

Simplified geometric representations of protein structures identify complementary interaction interfaces

“…Of primary interest for biological processes, is the assembly of large macromolecular machines. Using MorphProt, we explored the assembly of a large protein complex by examining our recently published Ceru+32/GFP‐17 protomer structure, 47 a synthetically engineered supercharged GFP 16‐mer (Figure 2D,E). These proteins were engineered to have oppositely charged variants of the normally monomeric green fluorescent proteins (GFP), which resulted in the assembly of a large, ordered macromolecular structure.…”

“…Of primary interest for biological processes, is the assembly of large macromolecular machines. Using MorphProt, we explored the assembly of a large protein complex by examining our recently published Ceru+32/GFP-17 protomer structure, 47 Figure S1).…”

Multi-modal engineering ofBstDNA polymerase for thermostability in ultra-fast LAMP reactions

“…Given that the villin headpiece likely assists with contacting nucleic acid substrates via positively charged patches, we sought to further improve this feature via the addition of excess positively charged amino acids (supercharging; (29)). We also anticipated that supercharging would improve the folding, solubility, and stability of the protein, by decreasing the propensity to aggregate, as we and others have previously demonstrated (30)(31)(32). We further anticipated that improvements gained via supercharging would be additive with substitutions introduced via machine-learning approaches, as supercharging targets additional biophysical mechanisms for stabilization, such as potentially improving interactions with the DNA substrate.…”