Comparison of the structural and dynamical properties of holo and apo bovine α-lactalbumin by NMR spectroscopy

In the presence of 0.5 M NaCl at pH 7.1, the Ca2+-free apo form of recombinant bovine x-lactalbumin (BLA) is sufficiently stabilised in its native state to give well-resolved NMR spectra at 20 degreesC. The H-1 and N-15 NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native hole protein. Large changes observed between the two forms of BLA are mainly limited to the Ca2+-binding region of the protein. These data suggest that Na+ stabilises the native apo state through the screening of repulsive negative charges, at the Ca2+-binding site or elsewhere, rather than by a specific interaction at the vacant Ca2+-binding site. The hydrogen exchange protection of residues in the Ca2+-binding loop and the C-helix is reduced in the apo form compared to that in the hole form. This indicates that the dynamic behaviour of this region of the protein is substantially increased in the absence of the bound Ca2+. Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the hole to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganisations of the structure in the vicinity of the Ca2+-binding site that are coupled to the intrinsic fluctuations in the protein structure. (C) 2001 Academic Press.