Impact of changes in buffer ionic concentration and mutations on a GH1 β-Glucosidase homodimer

Oligomerization is a key feature of protein function, with approximately 30% of proteins exhibiting this trait. The homodimeric form of proteins, such as the GH1 β-Glucosidase from Spodoptera frugiperda (Sfβgly), plays a significant role in enzyme activity. In this study, we investigate the homodimerization of Sfβgly, which forms a cyclic C2 dimer with a well-defined interface. Using size exclusion chromatography and SEC-MALS, we characterized the homodimerization behavior of Sfβgly at equilibrium conditions in different ionic concentrations of phosphate buffer. The dissociation constants ( K _D ) increase with decreasing ionic concentration, suggesting that the hydrophobic effect is central to homodimer formation. Site-directed mutagenesis of key residues at the dimer interface further elucidated the contributions of specific amino acid residues to dimer stability. Mutations affecting both, apolar and hydrogen bond-forming residues, significantly increased the K _D . However, mutations of hydrogen bond-forming residues caused a smaller K _D change than apolar residue mutations, suggesting that while the latter is the driving factor in the dimerization, the former may play a crucial role in guiding the monomers relative orientation. These findings enhance our understanding of protein oligomerization in GH1 β-Glucosidases and its implications for protein design and function.