Role of free Cys121 in stabilization of bovine beta-lactoglobulin B

Mixed disulfide derivatives of bovine beta-lactoglobulin (BLG) were studiedby circular dichroism (CD), gel-permeation HPLC and high- sensitivitydifferential scanning calorimetry (HS-DSC). It was shown that modificationof Cys121 with mercaptopropionic acid and mercaptoethanol does not affectthe secondary structure of BLG, but results instead in tertiary andquaternary structure changes. At neutral pH, the equilibriumdimer<==>monomer of modified beta- lactoglobulin is shifted towardsmonomeric form. In contrast to native BLG, thermal denaturation of modifiedbeta-lactoglobulin is fully reversible in neutral and acidic pH asdemonstrated by CD and HS-DSC measurements. Modification of Cys121 resultsin a significant decrease of transition temperature (-6 degrees C) andenthalpy (-106 kJ/mol) at pH 2.05 while unfolding heat capacity incrementremains unchanged. Thermal unfolding transitions of native and modifiedbeta-lactoglobulin at pH 2.05 are well approximated by a two-state modelsuggesting that no intermediate states appear after modification. Thedifference in Gibbs energy of denaturation between native and modifiedbeta- lactoglobulin, 8.5 kJ/mol at 37 degrees C and pH 2.05, does notdepend on the nature of the introduced group (charged or neutral). Computeranalysis of possible interactions involving Cys121 in a three- dimensionalstructure of beta-lactoglobulin revealed that the thiol group is too faraway from neighboring residues to form side-chain hydrogen bonds. Thissuggests that the sulfhydryl group of Cys121 may contribute to themaintenance of BLG tertiary structure via water mediated H-bonding.