High pressure NMR reveals a variety of fluctuating conformers in β-lactoglobulin

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine β-lactoglobulin at pH 2.0 and 36 °C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N′) of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the β-barrel, i.e. the hydrophobic core side (βF-H) (producing I2) and the non-core side (βB-E) (producing I1). At 1 bar the stability is higher for the core region (ΔG0=6.5(±2.0) kcal/mol) than for the non-core region (4.6(±1.3) kcal/mol), but at high pressure the stability is reversed due to a larger ΔV value of unfolding for the core region (90.0(±35.2) ml/mol) than that for the non-core region (57.4(±14.4) ml/mol), possibly due to an uneven distribution of cavities. The ΔG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of β-lactoglobulin, notably N, N′, I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.