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

High pressure H-1/N-15 two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta -lactoglobulin at pH 2.0 and 36 degreesC. Pressure dependencies of H-1 and N-15 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 H-1/N-15 cross peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta -barrel, i.e. the hydrophobic core side (betaF-H) (producing I-2) and the non-core side (betaB-E) (producing I-1). At 1 bar the stability is higher for the core region (DeltaG(0)=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 DeltaV 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 DeltaG(0) 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 beta -lactoglobulin, notably N, N', I-1, I-2, and the totally unfolded conformer U. Fluctuation of N to I-1 and I-2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure. (C) 2001 Academic Press.