A recombinant C121S mutant of bovine beta-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating

Biochemistry. 2004 May 25;43(20):6312-21. doi: 10.1021/bi0362469.


The lipocalin beta-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions. Each monomer contains two disulfide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability. We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S). Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH. The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 degrees C. Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion. Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid pH. We suggest that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anilino Naphthalenesulfonates / metabolism
  • Animals
  • Cattle
  • Crystallography, X-Ray
  • Dimerization
  • Disulfides / chemistry
  • Enzyme Stability
  • Fluorescent Dyes / metabolism
  • Hot Temperature*
  • Hydrogen-Ion Concentration
  • Lactoglobulins* / chemistry
  • Lactoglobulins* / genetics
  • Lactoglobulins* / metabolism
  • Milk / chemistry
  • Models, Molecular
  • Point Mutation*
  • Protein Conformation*
  • Protein Denaturation
  • Recombinant Proteins* / chemistry
  • Recombinant Proteins* / genetics
  • Recombinant Proteins* / metabolism
  • Reducing Agents / chemistry*


  • Anilino Naphthalenesulfonates
  • Disulfides
  • Fluorescent Dyes
  • Lactoglobulins
  • Recombinant Proteins
  • Reducing Agents
  • 1-anilino-8-naphthalenesulfonate