Altered ionization of the B13 Glu in insulin B9 and B10 mutants: a computational analysis

Protein Eng Des Sel. 2004 Jul;17(7):557-63. doi: 10.1093/protein/gzh066. Epub 2004 Aug 23.

Abstract

An experimentally determined pK(a) change of +2.50 units has been reported for the B13 Glu residue in a dimeric B9 Ser --> Asp insulin mutant relative to the native dimer. Poisson-Boltzmann electrostatics-based pK(a) calculations were performed to probe the effect of the B9 Ser --> Asp and B10 His --> Asp mutations on aggregation and the ionization behaviour of the B13 carboxylate. The method produced shifts of +2.64 and +2.45 units for the pK(a) shift of the two B13 residues in the B9 mutant dimer relative to the wild-type dimer, which is in good agreement with the experimental value (<6% error). The calculations also suggest that the reason neither mutant insulin can aggregate into hexamers is the resultant crowding of negatively charged groups in the central solvent channel on hexamer formation. In the wild-type insulin, binding of zinc ions by B10 His overcomes this problem, whereas in the B10 mutant this possibility is ruled out by the absence of the zinc binding site. A series of mutations are predicted to stabilize the medically relevant, monomeric form of insulin.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Crystallography, X-Ray
  • Dimerization
  • Glutamic Acid / chemistry*
  • Hydrogen-Ion Concentration
  • Insulin / chemistry*
  • Insulin / genetics*
  • Ions
  • Kinetics
  • Models, Molecular
  • Models, Theoretical
  • Molecular Sequence Data
  • Mutation*
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Software
  • Static Electricity
  • Swine
  • Zinc / chemistry

Substances

  • Insulin
  • Ions
  • Glutamic Acid
  • Zinc