pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and B

Abstract

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).

Electrostatics of folded and unfolded bovine β-lactoglobulin

“…Taken together, these findings indicate that the native BLG structure is more stable at acidic than at alkaline pH, in agreement with our previous results with DGGE at varying pH (Beringhelli et al 2002) and with other literature data (D'Alfonso et al 2002;Fogolari et al 2000;Rochu et al 1999;Shalabi and Fox 1982).…”

“…These details are also observed with all runs in media at pH [6.1 (Beringhelli et al 2002). The overall trend is similar for BLG isoform A (Fig.…”

“…This suggests that the folding-unfolding transition may have peculiar features at acidic pH. However, DGGE migration even at lower pH, 3.8, produces a sigmoidal (left) curve (Beringhelli et al 2002). Similar contrasting findings are observed also for BLG isoform A (Fig.…”

“…Creighton's group has not only laid the theoretical basis for DGGE (Creighton 1979(Creighton , 1980, but also defined the optimal experimental conditions for the runs, including the selection of a few low conductivity, high buffering capacity media of different pH [3.8, 4.8, 6.1, 7.4, 8.7 (Goldenberg 1989)], which are since in common use, including by our lab (Beringhelli et al 2002;Gianazza et al 1997). Running DGGE on BLG isoform B at pH 6.1 across a 0-8 M urea gradient results in the denaturation curve shown in the rightmost panel in the lower row of Fig.…”

“…Sometime ago, some of us published a comprehensive assessment of the pH-dependence of several structural and functional features of BLG (Beringhelli et al 2002). As part of Supporting information ( Figure S2), we posted the patterns of the protein in denaturant gradient gel electrophoresis (DGGE), i.e., after migration across a transverse urea gradient in buffers at different pH (Creighton 1979).…”