Conformational analysis by nuclear magnetic resonance: insulin.

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).