Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

Abstract

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

Figure 1

Position 76 in RNase Sa and neighboring turn residues (Ala75, Gln77, and Glu78). The side chain of Thr76 is 2.5% hyper-exposed to solvent as determined by pfis. The figure was generated using the Swiss-Pdb Viewer program.

Figure 2

Effect of ammonium sulfate concentration on the melting temperature of T76D Sa in 50 mM diglycine buffer at pH 9.0.

Figure 3

RNase Sa solubility as a function of ammonium sulfate concentration in 50 mM sodium acetate buffer at pH 4.25.

Figure 4

Solubility curves as a function of ammonium sulfate concentration at pH 4.25 for the aspartic acid (circles), alanine (squares), and tyrosine (diamonds) variants.

Figure 5

Hydrophobicity values as reported by Fauchere and Pliska plotted against normalized pH 4.25 solubility values. Solubility values were normalized to the solubility value of the glycine variant. (a) Correlation for the hydrophilic variants. (b) Correlation for hydrophobic variants.

Figure 5

Hydrophobicity values as reported by Fauchere and Pliska plotted against normalized pH 4.25 solubility values. Solubility values were normalized to the solubility value of the glycine variant. (a) Correlation for the hydrophilic variants. (b) Correlation for hydrophobic variants.