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 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (K(D), ΔH, ΔG, and ΔS) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed ΔH 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 β subunit, which is known to bind to the ILPR G-quadruplex DNA.
Department of Chemistry and Biochemistry, Calvin College , 3201 Burton Street Southeast, Grand Rapids, Michigan 49546, United States.
Christine M
CM
Timmer
Nicole L
NL
Michmerhuizen
Amanda B
AB
Witte
Margaret
M
Van Winkle
Dejian
D
Zhou
Kumar
K
Sinniah
D000818:Animals; D002021:Buffers; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D019277:Entropy; D054856:G-Quadruplexes; D006531:HEPES / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry*; D016014:Linear Models; D008968:Molecular Conformation; D010710:Phosphates / Q000737:chemistry; D011522:Protons; D013696:Temperature; D013816:Thermodynamics; D014732:Vibration
D002021:Buffers; D007328:Insulin; D010710:Phosphates; D011522:Protons; D004247:DNA; D006531:HEPES