An isothermal titration and differential scanning calorimetry study of the G-quadruplex DNA-insulin interaction

The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 oC upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (KD, H, G and S) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which and is mainly driven by entropic factors at below 25 °C but by enthalpic factors terms at above 30 °C. The interaction was also examined in several different buffers and results showed that observed H is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin’s beta subunit, which is known to bind to the ILPR G-quadruplex DNA.