The Gp2 domain is a 45 amino-acid scaffold that has been evolved for specific, high-affinity binding towards multiple targets and was proven useful in molecular imaging and biological antagonism. It was hypothesized that Gp2 may benefit from increased hydrophilicity for improved physiological distribution as well as for physicochemical robustness. We identified seven exposed hydrophobic sites for hydrophilic mutations and experimentally evaluated single mutants, which yielded six mutations that do not substantially hinder expression, binding affinity or specificity (to epidermal growth factor receptor), and thermal stability. Eight combinations of these mutations improved hydrophilicity relative to the parental Gp2 clone as assessed by reverse-phase high-performance liquid chromatography (p < 0.05). Secondary structures and refolding abilities of the selected single mutants and all multimutants were unchanged relative to the parental ligand. A variant with five hydrophobic-to-hydrophilic mutations was identified with enhanced solubility as well as reasonable binding affinity (K-d = 53-63 nM), recombinant yield (1.3 +/- 0.8 mg/L), and thermal stability (T-m = 53 +/- 3 degrees C). An alternative variant with a cluster of three leucine-to-hydrophilic mutations was identified with increased solubility, nominally increased binding affinity (K-d = 13-28 nM) and reasonable thermal stability (T-m = 54.0 +/- 0.6 degrees C) but reduced yield (0.4 +/- 0.3 mg/L). In addition, a >= 7 degrees C increase in the midpoint of thermal denaturation was observed in one of the single mutants (T21N). These mutants highlight the physicochemical tradeoffs associated with hydrophobic-to-hydrophilic mutation within a small protein, improve the solubility and hydrophilicity of an existent molecular imaging probe, and provide a more hydrophilic starting point for discovery of new Gp2 ligands towards additional targets.