Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

Abstract

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…In solutions at pH 5 with 125 mM sodium chloride, the k D value for 5E is much greater than either the 5K or 5R mutant (see Table 2). The increased solubility observed here of negative versus positive charged groups has also been deduced from mutation studies of Ribonuclease SA [89] and from the salting-out behaviour for a series of seven proteins [44].…”

“…To avoid increasing protein charge anisotropy, charged mutations should carry the same sign as the net charge on the corresponding protein scaffold [31,100]. Deconvoluting between these competing charge effects has led to confusion over whether negatively charged mutations are more effective than positively charged mutations [44,89]. Further, solubilizing effects of charged mutations are also specific to the chemical nature of the residue concerned.…”

One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen

“…Of the 18 mutations, 15 occurred on the surface, of which 8 improved surface polarity, and an additional 2 (Lys312Asn and Lys316Asn) eliminated a homogenous positively charged patch. Indeed, such patches have been associated with aggregation and poor stability (24,25). Furthermore, PfRH5 comprises a mostly helical backbone (75% of the sequence), and seven mutations increased helix-forming propensity relative to PfRH5.…”

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

“…The mutations identified mainly affected solvent-exposed residues (V26I, Q45H, L193M, T339I; Fig. 5C), which may be indicative of their involvement in protein stabilization and folding efficiency (25)(26)(27). One mutation (N122S) is located close to the catalytic residue D118, in a region previously shown to be involved in substrate specificity in amino acid dehydrogenases (28).…”