Electrostatics of folded and unfolded bovine β-lactoglobulin

Abstract

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine β-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5-9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pKa value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pKa value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.

Solvatochromism as a new tool to distinguish structurally similar compounds

“…It should be mentioned that no significant differences between the polypeptide structures in RNase A and B are detected by other techniques, such as X-ray analysis 28. The results for the transferrin’s are in agreement with the results obtained by partitioning in aqueous two-phase systems29, whereas the results for the β-lactoglobulins A and B isoforms are in agreement with the results obtained by partitioning in aqueous two-phase systems30, gradient chromatofocusing31 and electrophoretic, spectroscopic, and computational studies of the isoforms32, all used as different approaches for identifying structurally similar proteins.…”

Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes

“…Consistent with what was discussed above, both salts and nonionic chaotropes, such as urea, also may promote dissociation of the BLG dimer into monomers at pH values close to neutrality. Whereas the effects of lyotropic salts may just be due to the competitive screening of ionic interactions such as those involved in dimerization at neutral pH, the effects of lipotropic salts and of chaotropes are more extensive, and appear to involve regions of the protein other than the contacts between oppositely charged side chains reportedly involved in dimerization [ 33 ].…”

“…As anticipated in the Introduction, in native BLG, this thiol is buried underneath the main C-terminus alpha-helix. Since movement of the alpha-helix away from the barreled body of BLG is known to be among the earliest and reversible steps of BLG unfolding [ 33 , 34 ], reactivity of Cys121 can be used to monitor this particular conformational change.…”

“…As anticipated in the Introduction, in native BLG, this thiol is buried underneath the main C-terminus alpha-helix. Since movement of the alpha-helix away from the barreled body of BLG is known to be among the earliest and reversible steps of BLG unfolding [33,34], reactivity of Cys121 can be used to monitor this particular conformational change. At ionic strength values below unity, all salts appear to have some stabilizing effect on BLG, as inferred by the increase in T m (the temperature at which 50% of the thiols are exposed) (Figure 2A).…”

Exposure of thiol groups in the heat-induced denaturation of β-lactoglobulin

“…While these results seem to suggest that the protonation state of the free cysteine plays an important role in the aggregation process, it is unclear how to link these results directly to experiments, as the pK a of the buried cysteine is probably very different from the standard pK a of a cysteine in solution. Recent work by Eberini et al [29] suggests that the pK a of C121 could go from 10.56 in the native state to 7.28 in the unfolded state. In this light it seems unlikely that the free cysteine is deprotonated in the native state.…”