Mitigating Alkaline Instability Induced by Tyrosine–Tyrosine Repulsion in an FcγRIIIa-Binding Protein Through Phenylalanine Substitution

Proteins are essential components in biotechnological and biopharmaceutical applications; however, their structural instability under alkaline conditions presents significant limitations. High-pH environments, such as chromatographic clean-in-place (CIP) protocols, frequently cause protein degradation and loss of biological activity. Current strategies for engineering alkali-stable proteins include rational design approaches targeting deamidation-susceptible residues, surface charge optimization, and enzyme extraction from alkaliphilic organisms. However, the fundamental principles governing alkaline stability remain poorly understood. In this study, we investigated alkaline stability mechanisms in Fc gamma receptor IIIa, a critical immune effector protein with applications in antibody purification and glycoform analysis. Systematic mutagenesis identified a tyrosine-to-phenylalanine substitution at position 59 that significantly enhanced protein stability during alkaline CIP exposure while retaining substantial IgG binding activity. Structural and biophysical characterizations revealed that this substitution prevents the deprotonation of tyrosine that occurs at alkaline pH, thereby mitigating destabilizing electrostatic repulsion within the protein structure. Our findings support a model in which targeted aromatic substitution enhances alkaline stability without severely compromising protein function and provide mechanistic insight into the contribution of buried tyrosine ionization to alkaline instability in FcγRIIIa.