Proton fourier transform NMR studies of insulin: coordination of calcium to the Glu(B13) site drives hexamer assembly and induces a conformation change

Abstract

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.

The influence of ionic strength and pH on the aggregation properties of zinc‐free insulin studied by static and dynamic laser light scattering

“…where the subscript i is the aggregation number, i.e., M i represents an oligomer consisting of i monomeric units and Ki, is related to Gibbs free energy by AGij = -RT In Kij = AG, + AGG (12) In the latter relation the superscripts plus and minus indicate the positive and negative part of Gibbs free energy.…”

“…After an equilibrium distribution has formed according to the equations of the association model, there is in principle a virial effect, on each equilibrium oligomer present, from the interaction potential described by Eq. (12). If one assumes that the attractive part is of enthalpic origin, one can approximate it with a square well potential3' with a width to be determined by a fit.…”

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

“…The association/dissociation of the protein hormone insulin has been investigated by several experimental techniqes, such as equilibrium sedimentation (Jeffrey and Coates 1966 a, b), static light scattering (Doty et al 1952;Steiner 1951;Bohidar and Geissler 1984), concentration Correspondence to: J. Skov Pedersen difference UV spectroscopy (Lord et al 1973;Strazza et al 1985), rapid kinetics (Coffman and Dunn 1988), circular dichroism (Goldman and Carpenter 1974;Pocket and Biswas 1981), osmotic pressure (Hansen 1991), and nuclear magnetic resonance (Palmieri et al 1988;Roy et al 1990; Kadima et al 1992). The monomeric species of insulin has a molecular weight of 5800 Daltons.…”

Construction of Insulin 18‐mer Nanoassemblies Driven by Coordination to Iron(II) and Zinc(II) Ions at Distinct Sites

“…[11] De novo design has combined 2,2'-bipyridine (bipy) with anative-like Cu-binding site in athree-helix bundle metalloprotein. [14] Hexamers with Cd II ,Co II ,Ni II ,Cu II ,Mn II ,Fe II , and Co III ions were observed. [13] Human insulin (HI) is aprotein crucial for regulating the blood glucose level.…”

“…[5] Another approach is to use native metalion-coordinating amino acids to link protein surfaces.Exam-ples include model systems with cytochrome ct ogether with proteins such as ferritin, [6] T4 lysozyme and maltose-binding protein, [7] them onomeric Rab4-binding domain of rabenosyn, [8] and Ni II -directed formation of glutathione Stransferase (GST) nanorings. [14] Hexamers with Cd II ,Co II ,Ni II ,Cu II ,Mn II ,Fe II , and Co III ions were observed. [11] De novo design has combined 2,2'-bipyridine (bipy) with anative-like Cu-binding site in athree-helix bundle metalloprotein.…”