Role of free Cys121 in stabilization of bovine beta-lactoglobulin B.
Mixed disulfide derivatives of bovine beta-lactoglobulin (BLG) were studied by circular dichroism (CD), gel-permeation HPLC and high-sensitivity differential scanning calorimetry (HS-DSC). It was shown that modification of Cys121 with mercaptopropionic acid and mercaptoethanol does not affect the secondary structure of BLG, but results instead in tertiary and quaternary structure changes. At neutral pH, the equilibrium dimer<==>monomer of modified beta-lactoglobulin is shifted towards monomeric form. In contrast to native BLG, thermal denaturation of modified beta-lactoglobulin is fully reversible in neutral and acidic pH as demonstrated by CD and HS-DSC measurements. Modification of Cys121 results in a significant decrease of transition temperature (-6 degrees C) and enthalpy (-106 kJ/mol) at pH 2.05 while unfolding heat capacity increment remains unchanged. Thermal unfolding transitions of native and modified beta-lactoglobulin at pH 2.05 are well approximated by a two-state model suggesting that no intermediate states appear after modification. The difference in Gibbs energy of denaturation between native and modified beta-lactoglobulin, 8.5 kJ/mol at 37 degrees C and pH 2.05, does not depend on the nature of the introduced group (charged or neutral). Computer analysis of possible interactions involving Cys121 in a three-dimensional structure of beta-lactoglobulin revealed that the thiol group is too far away from neighboring residues to form side-chain hydrogen bonds. This suggests that the sulfhydryl group of Cys121 may contribute to the maintenance of BLG tertiary structure via water mediated H-bonding.
Burova
T V
TV
Institute of Biochemical Physics, Russian Academy of Sciences, Moscow.
Choiset
Y
Y
Tran
V
V
Haertlé
T
T
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D007782:Lactoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D007782:Lactoglobulins; D003545:Cysteine