Effects of Nucleotides on the Denaturation of F Actin: A Differential Scanning Calorimetry and FTIR Spectroscopy Study

Abstract

We have utilized DSC and high pressure FTIR spectroscopy to study the specificity and mechanism by which ATP protects actin against heat and pressure denaturation. Analysis of the thermograms shows that ATP raises the transition temperature Tm for actin from 69.6 to 75.8°C, and the calorimetric enthalpy, ΔH, from 680 to 990 kJ/mole. Moreover, the peak becomes sharper indicating a more cooperative process. Among the other nucleotide triphosphates, only UTP increases the Tm by 2.5°C, whereas GTP and CTP have negligeable effects; ADP and AMP are less active, increasing the Tm by 2.1 and 1.6°C, respectively. Therefore, γ phosphate plays a key role in this protection, but its hydrolysis is not implicated since the nonhydrolysable analogue of ATP, ATP-PNP have the same activity as ATP. FTIR spectroscopy demonstrates that ATP also protects actin against high pressure denaturation. Analysis of the amide I band during the increase in pressure clearly illustrates that ATP protects particularly a region rich in β-sheets of the actin molecule.