Amino Acid Contribution to Protein Solubility: Asp, Glu, and Ser Contribute more Favorably than the other Hydrophilic Amino Acids in RNase Sa

Abstract

SUMMARYPoor 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 twenty amino acids to protein solubility has not been done. Here, twenty 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 β-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.

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.…”

Antibodyomics: bioinformatics technologies for understanding B‐cell immunity to HIV‐1

“…As hydrophobic interactions are often a major contributor to binding affinity, hydrophobic mutations at different surface locations can identify variants with enhanced potency 103, 104. Similarly, hydrophilic mutations and N‐ linked glycan can identify variants with enhanced solubility 105, 106. Moreover, N ‐linked glycan can modulate autoreactivity and also inform antibody proximity to antigen 107.…”

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

“…The pH dependence of the thermal unfolding of H2A-H2B and H3-H4 has been reported, although at lower ionic strengths. 41,42 Between pH 4.5 and 5.5, the mid-point of the thermal unfolding transition, T M , for the H3-H4 heterodimer was [50][51][52][53][54][55][56][57][58][59][60] C, depending on the protein concentration, while the T M of H2A-H2B was 10-15 C lower. Thus, H3-H4 is more stable, but forms fibrils much more readily than H2A-H2B or the individual H2A and H2B monomers which are only partially folded even under stabilizing conditions.…”

“…55,57 A protein's electrostatic properties influence solubility, but there are additional aspects of protein sequence and structure that dictate solubility. 56 Despite the acknowledged importance, there have been relatively few studies directly relating solubility to fibrillation propensity. Using the model system of RNAse Sa and mutants with varying charge, it was shown the pH dependence of fibril formation correlates with changes in the protein's isoelectric point, pI, where it is least soluble.…”