PEDS Advance Access originally published online on August 23, 2004
Protein Engineering Design and Selection 2004 17(7):557-563; doi:10.1093/protein/gzh066
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COMMUNICATION |
Altered ionization of the B13 Glu in insulin B9 and B10 mutants: a computational analysis
1Department of Biology and 4Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, 2Department of Biomolecular Sciences, UMIST, P.O. Box 88, Manchester M60 1QD, 5Protein Structure Division, NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK and 6Bioinformatics Institute, 30 Biopolis Way, #07-01 Matrix, Singapore 138671
3 To whom correspondence should be addressed (at the UMIST address for R.B.G.). E-mail: r.greaves{at}umist.ac.uk or chandra{at}bill.a-star.edu.sg
An experimentally determined pKa 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 pKa 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 pKa 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.
Received April 8, 2004; revised July 28, 2004; accepted August 1, 2004.
Edited by Bruce Tidor