Structural transition in the metal-free hexamer of protein-engineered [B13 Gln]insulin.

For hexamer formation of native insulin the repulsive potential of six B13 Glu carboxylate groups coming together in the centre is overcome by zinc binding to B10 His. Substitution of Gln for Glu in position B13 by site-directed mutagenesis, i.e. replacement of the repelling carboxylates by amide groups, which are offering H-bonding potential, enhances association and allows a metal-free hexamer to form. Merely upon addition of zinc ions this hexamer undergoes the T6----T3R3 respectively T6----R6 structural transition which in the native 2Zn insulin hexamer is inducible only by additives like inorganic anions or phenolic compounds. [B13 Gln]Insulin hexamers are transformed by phenolic compounds, but not by anions, even in the absence of any metal. The structural transformation of insulin can thus be brought about in two ways: By inorganic ions with the zinc ions as their points of attack, which preexist in the nontransformed hexamer, and by phenol, for which the binding sites close to the B5 histidines come into existence only with the transformation. Therefore transformed and non-transformed hexamers, i.e. molecules with helical and extended B chain N-terminus, must be related in a dynamic equilibrium. Phenol acts as a wedge jamming the structure in the transformed state and trapping the zinc ions. Combination of transformed 2Zn[B13 Gln]insulin and metal-free native insulin in the absence of additives results in a redistribution of the zinc ions in favour of native insulin which is an outcome of the dynamic equilibrium and also demonstrates an influence of B13 charge on metal binding affinity. Transformation of a single subunit in a hexamer would lead to bad contacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenol-promoted structural transformation of insulin in solution.

Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2 Zn----4 Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of Zn2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni2 which is unable to replace Zn2 in 4 Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2 Zn----4 Zn transformation. 2 Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4 Zn conformation. Phenol, o- and p-cresol, m-nitrophenol, Nipagin M and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation.