Supercharging Proteins Can Impart Unusual Resilience

Abstract

Protein aggregation, a well-known culprit in human disease, 1,2 is also a major problem facing the use of proteins as therapeutic or diagnostic agents. 3,4 Insights into the protein aggregation problem have been garnered from the study of natural proteins, where a relationship between solubility and net charge has been noted. For example, it is known that proteins are least soluble at their isoelectric point, where they bear a net charge of zero. 5 More recently, small differences in net charge ((3 charge units) have been shown to predict aggregation tendencies among peptide variants. 6 In addition, intrinsically disordered proteins, 7,8 a class of proteins that are largely unfolded in the cell but that do not lead to pathological aggregation, tend to have large net charges. 9, 10 We speculated that the relationship between net charge and aggregation resistance might also be applicable to globular proteins, which can aggregate via partial unfolding induced by thermal agitation, chemical treatment, or conformational breathing. Recent evidence that some proteins can tolerate significant changes in net charge (for example, the finding that carbonic anhydrase retains activity after exhaustive acetylation of its surface lysines 11 ) encouraged us to test the hypothesis that the solubility and aggregation resistance of some proteins might be significantly enhanced, without abolishing their folding or function, by extensively mutating their surfaces to dramatically increase their net charge, a process we refer to as "supercharging".We began with green fluorescent protein (GFP), an easily assayed protein that undergoes chromophore maturation and becomes fluorescent only when folded correctly. To minimize the possibility that our GFP was unusually delicate and therefore unusually easy to improve, we used a starting GFP (stGFP) based on the stateof-the-art GFP variant called "superfolder", which has been highly optimized for folding robustness and resistance to denaturants. 12 The net charge of the stGFP is -7, similar to that of wild-type GFP (-8). To create a superpositive variant of GFP, we identified 29 positions in the crystal structure that were highly solvent-exposed and mutated these to positively charged amino acids (Lys and Arg), yielding a design with a theoretical net charge of +36 ( Figure 1 and Supporting Information). Genes encoding stGFP and GFP-(+36) were constructed and expressed in E. coli, and both genes yielded intensely green fluorescent bacteria. Following protein purification, the fluorescence properties of GFP(+36) were measured and found to be very similar to those of stGFP (Supporting Information).Encouraged by this finding, we produced and characterized additional supercharged GFPs having net charges of +48, -25, and -30, all of which were also found to have stGFP-like fluorescence (Figures 2a and S1). All supercharged GFP variants exhibited circular dichroism spectra similar to that of stGFP, indicating that the proteins have similar secondary structure content (Figure 2b). The thermodynamic s...

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

“…In water-soluble globular proteins, large numbers of surface residues can be mutated without disrupting protein stability as long as surface hydrophilicity is maintained (55), and hydrophobic α-helical membrane protein surfaces have been shown to be similarly tolerant. It had been suggested that the same was true for β-barrel membrane protein surfaces (44), a prediction that makes intuitive sense based on the favorable energetic contributions made by lipid-facing hydrophobic residues (56), and known evolutionarily allowed mutation patterns (57).…”

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

“…S1) were designed based on super-charged GFPs (54). All three were synthesized and cloned into pET30a(þ) vectors (GeneCust), expressed as His-GFP fusion proteins in Escherichia coli BL21(DE3), and purified according to previously established protocols (52,54), with minor modifications. Full details are provided in the Supporting Material.…”

RNA-mediated chaperone type for de novo protein folding

“…As an alternative, the net charge of a given protein is closely correlated with protein solubility. 35 In addition, the net charge of small and large tags is also important for their solubilizing ability. 36,37 It was suggested that mechanistically the electrostatic repulsions between proteins could prevent protein aggregation.…”