Binding a C12-appended rhenium-(Bispyridine) carbonyl complex to β-Lactoglobulin: Effects of pH & cysteine modification on calyx affinity

Due to its commercial availability and well-defined structure, the interaction between bovine protein β-lactoglobulin (βLG) and a wide variety of non-native ligands — including transition metal complexes — has been explored, but its application as an artificial metalloenzyme scaffold is limited. This protein is hypothesized to transport fatty acids and other nutrients during juvenile development, and it binds hydrophobic ligands inside a binding pocket constructed upon an 8-stranded β-barrel, called the ‘calyx’. Herein, we compare the binding behavior of two rhenium(anthracene-bispyridine) (‘Anth-py₂’) tricarbonyl complexes, one with a 12‑carbon chain appended to the ligand scaffold (‘^C12Anth-py₂’) to βLG. We investigate (i) how calyx-binding specificity is affected by pH (which controls βLG structure at the entrance to the calyx) and (ii) modification of a free cysteine residue located in a putative second binding site of βLG (SMe-βLG). The binding affinities of [Re(^C12Anth-py₂)(CO)₃(solv)]⁺ (ReC₁₂) and [Re(Anth-py₂)(CO)₃(solv)]⁺ (ReCH) for βLG at pH 7.3 were similar at 36 ± 2 μM and 43 ± 1 μM, respectively. The K_D of ReC₁₂ decreased by ∼13 μM at pH 6.1 due to a well-known conformational change (Tanford transition) at the entrance to the calyx; the K_D value was not significantly affected by Cys121 modification, indicating β-barrel calyx binding specificity. In contrast, ReCH experienced a decrease in K_D in response to blocking the second binding (SMe-βLG), but was also unaffected by pH. The results show an increase in binding affinity and specificity as a result of targeted ligand design and utilization of native protein characteristics. The findings will inform and improve the design of future βLG-derived ArMs.