SOLUTION STRUCTURE OF AN ENGINEERED INSULIN MONOMER AT NEUTRAL PH

Insulin circulates in the bloodstream and binds to its specific cell-s urface receptor as a 5808 Da monomeric species. However, studies of th e monomer structure and dynamics in solution are severely limited by i nsulin self-association into dimers and higher oligomers. In the prese nt work we use site-directed mutagenesis of the dimer- and hexamer-for ming surfaces to yield the first insulin species amenable for structur e determination at neutral pH by nuclear magnetic resonance (NMR) spec troscopy. The preferred insulin mutant, i.e., (B1, B10, B16, B27) Glu, des-B30 insulin retains 47% biological potency and remains monomeric at millimolar concentrations in aqueous solution at pH 6.5-7.5 as judg ed by NMR and near-UV circular dichroism (CD) spectroscopy. From a ser ies of 2D H-1-NMR spectra collected at pH 6.5 and 34 degrees C, the ma jority of the resonances are assigned to specific residues in the sequ ence, and nuclear Overhauser enhancement (NOE) cross-peaks are identif ied. NOE-derived distance restraints in conjunction with torsion restr aints based on measured coupling constants, (3)J(HNH alpha), are used for structure calculations using the hybrid method of distance geometr y and simulated annealing. The calculated structures show that the maj or part of the insulin mutant is structurally well defined with an ave rage root mean square (rms) deviation between the 25 calculated struct ures and the mean coordinates of 0.66 Angstrom for backbone atoms (A2- A19 and B4-B26) and 1.31 Angstrom for all backbone atoms. The A-chain consists of two antiparallel helices, A2-A7 and A12-A19, connected by a loop. The B-chain contains a loop region (B1-B8), an alpha-helix (B9 -B19), and a type I turn (B20-B23) and terminates as an extended stran d (B24-B29). The B1-B4 and B27-B29 regions are disordered in solution. The structure is generally similar to crystal structures and resemble s a crystalline T-state more than an R-state in the sense that the B-c hain helix is confined to residues B9-B19.