Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogenexchange measurements

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging t o the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, r espectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hy drophobic surface patch provides stabilising interactions between the barre l and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the pre sence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasin g amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting th e presence of slow exchange between native and unfolded protein. Hydrogen e xchange measurements at different urea concentrations were performed in ord er to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibriu m energetic for the global transition and the local fluctuations. Most of t he residues converge to define a common isotherm identifying a unique coope rative folding unit, encompassing all the strands, except strand beta(1), a nd the terminal region of the helix. The amides that do not join the same g lobal unfolding isotherm are characterised by low Delta G(op)(H2O) and espe cially by low m values, indicating that they are already substantially expo sed in the native state. A two-state unfolding model N reversible arrow U i s therefore proposed for this rather big protein, in agreement with CD data . Renaturation studies show that the unfolding mechanism is reversible up t o 6 M urea and suggest a similar unfolding and refolding pathway. Present r esults are discussed in Light of the hypothesis of an alpha --> beta transi tion proposed for bovine beta-LG refolding. (C) 1999 Academic Press.