The protein dynamics of bovine and caprine β-lactoglobulin differ as a function of pH

The properties of milk proteins differ between mammalian species. β-Lactoglobulin (βlg) proteins from caprine and bovine milk are sequentially and structurally highly similar, yet their physicochemical properties differ, particularly in response to pH. To resolve this conundrum, we compared the dynamics of both the monomeric and dimeric states for each homologue at pH 6.9 and 7.5 using hydrogen/deuterium exchange experiments. At pH 7.5, the rate of exchange is similar across both homologues, but at pH 6.9 the dimeric states of the bovine βlg B variant homologue have significantly more conformational flexibility compared with caprine βlg. Molecular dynamics simulations provide a mechanistic rationale for the experimental observations, revealing that variant-specific substitutions encode different conformational ensembles with different dynamic properties consistent with the hydrogen/deuterium exchange experiments. Understanding the dynamic differences across βlg homologues is essential to understand the different responses of these milks to processing, human digestion, and differences in immunogenicity.