HIGH-RESOLUTION STRUCTURE OF AN ENGINEERED BIOLOGICALLY POTENT INSULIN MONOMER, B16 TYR -] HIS, AS DETERMINED BY NUCLEAR-MAGNETIC-RESONANCESPECTROSCOPY

Site-directed mutagenesis is used in conjunction with H-1 nuclear magn etic resonance (NMR) and circular dichroism (CD) spectroscopy in order to find an insulin species amenable for structure determination in aq ueous solution by NMR spectroscopy. A successful candidate in this res pect, i.e., B16 Tyr --> His mutant insulin, is identified and selected for detailed characterization by two-dimensional H-1 NMR. This mutant species retains 43% biological potency and native folding stability, but in contrast to human insulin it remains monomeric at millimolar co ncentration in aqueous solution at pH 2.4. The resulting homogeneous s ample allows high-quality 2D NMR spectra to be recorded. The NMR studi es result in an almost complete assignment of the H-1 resonance signal s as well as identification of NOE cross peaks. NOE-derived distance r estraints in conjunction with torsion restraints based on measured cou pling constants, (3)J(HNH alpha), are used for structure calculations using the hybrid method of distance geometry and simulated annealing. The calculated structures show that the major part of the insulin mono mer is structurally well-defined with an average rms deviation between the 20 calculated structures and the mean coordinates of 0.89 Angstro m for all backbone atoms, 0.46 Angstrom for backbone atoms (A2-A19 and B4-B28), and 1.30 Angstrom for all heavy atoms. The structure of the A-chain is composed of two helices from A2 to A7 and from A12 to A19 c onnected by a short extended strand. The B-chain consists of a loop, B 1-B8, an alpha-helix, B9-B19, a beta-turn, B20-B23, and an extended st rand from B24 to B30. Residues A1-A6, B1-B8, and B28-B30 are less well defined than the rest of the polypeptide. The solution structure rese mbles structures in crystals, in particular, molecule 1 of T-6 pig ins ulin.