Protamine and Zinc Binding-Induced Conformational Effects on Insulin Analogs: An Integrated Experimental and Computational Study

Abstract

Insulin and its modified analogs play a critical role in the development and function of various insulin formulations, particularly concerning insulin’s time-dependent action. Given that zinc promotes the formation of insulin hexamer, the most stable form, we aim to investigate the conformational dynamics of insulin analogs, specifically lispro and aspart, in both the absence and presence of zinc. Additionally, we seek to study their interactions with protamine, a key component of insulin formulations. Our experimental findings indicate that the zinc-bound states of lispro and aspart are more stable compared to their zinc-free forms. Terpyridine-induced dissociation experiments revealed that zinc-lispro dissociates more rapidly than zinc-aspart, suggesting that zinc-lispro forms a less tightly assembled hexamer than zinc-aspart. Atomic force microscopy (AFM) results show that zinc-lispro forms a more heterogeneous assembly as evidenced by broader distribution curve. In contrast, zinc-aspart maintains a more consistent hexameric structure, resembling the behavior of native insulin. This observation is further supported by circular dichroism (CD) spectroscopy, which showed a lower helical content in zinc-lispro compared to zinc-aspart. Molecular dynamics (MD) simulations successfully corroborate these experimental results, providing a comprehensive understanding of the structural and dynamic differences. Regarding the effect of protamine, our isothermal titration calorimetry (ITC) and fluorescence studies indicate that protamine binds more strongly to more stable zinc-aspart compared to Zn-lispro. These findings indicate that protamine preferentially binds to the more stable hexameric form. ANS binding studies reveal that both lispro and aspart undergo conformational changes in the presence of protamine, leading to the exposure of their hydrophobic residues. Furthermore, hydrogen bond analysis from MD simulations indicates that zinc-aspart forms more hydrogen bonds with protamine than zinc-lispro. This is likely due to the higher polarity of the aspartic acid residue, which enhances its complexation with protamine.