Hidden protein states with partially unfolded regions can have a functional relevance and may also contribute to an uncontrolled self-association of proteins leading to pathological conditions. Human ileal bile acid-binding protein (hI-BABP), a member of the family of intracellular lipid-binding proteins (iLBPs), plays a key role in the transcellular trafficking and metabolic targeting of bile salts. Disorder-order transitions and sparsely populated hidden states in hI-BABP are thought to be key elements of ligand recognition, with implications for bile salt recycling and bile salt-mediated signaling events. To improve our understanding of the structural determinants of hI-BABP stability, high pressure NMR was used to probe local packing interactions and characterize the folding/unfolding process. Heterogeneity in pressure response, revealed by residue-specific analysis of chemical shift and intensity changes, indicates a deviation from two-state unfolding. Four specific protein regions such as the N-terminal β-strand, the helical cap, a hydrophobic cluster at the bottom of the β-barrel, and segments of the HIJ-region have been found to exhibit a pressure response differing significantly from the rest of the protein. Our analysis further shows that a low-populated higher-energy state, inferred by NMR relaxation dispersion measurements, becomes less distinct from the ground state at high pressure, corroborating the hypothesis that conformational exchange between a closed and a more open EFGH-region, which mediates ligand entry, is related to a partial unfolding of the protein. A pathway of destabilization induced by high hydrostatic pressure is proposed and discussed in relation to the response of iLBPs to other stress conditions.
Teski et al. (2026) studied this question.