Structural details of Asp(B9) human insulin at low pH from two-dimensional NMR titration studies.

A titration study of the dimeric Asp(B9) mutant of human insulin was performed using two-dimensional NMR spectroscopy. Based on 10 NOESY spectra recorded in the pH range 1.73-3.93, the pKa values of the seven carboxyl groups in the mutant were determined, and the titration shifts of 46 pH-dependent protons in non-ionizable groups were investigated. Further, the pKa values of the two histidine imidazole rings were determined from a series of 1D spectra recorded in the pH range 6.65-10.0. The titration shifts of all pH-dependent protons were analyzed by a nonlinear least-squares fitting procedure, using an equation that describes a one-step titration. Also the pH dependence of the exchange rate of the amide proton of Phe(B24) was determined in the applied pH range. On the basis of the experimental results, it is concluded that the Asp(B9) residue forms an N-cap of the B-chain alpha-helix through an interaction between the side-chain carboxyl group of the residue and the dipole of the helix. Further, the titration data show that salt bridges are established between Glu(B13) and His(B10) and between Asn(A21) and Arg(B22) at pH values, where the interacting groups are ionized, and that a hydrogen bond exists between the amide proton of Val(A3) and the C-terminal carboxyl group of Thr(B30). Most surprisingly, the data analysis shows that the Asp(B9) insulin exists as a dimer throughout the investigated pH range, that is, also at pH values where there is a substantial negative charge repulsion in the monomer-monomer interface of the dimer.