Thermodynamics of the interaction between dengue virus NS3 helicase and single stranded RNA: temperature and natural ligands effects.

J. JEREMIAS INCICCO; ANDREA V. GAMARNIK; LEILA A. CABABIE; EVELYN B. MIKKELSEN; F. LUIS GONZÁLEZ FLECHA; SERGIO B. KAUFMAN; LEOPOLDO G. GEBHARD

La Plata

Congreso; XLVII Reunión Anual SAB; 2018

Sociedad Argentina de Biofísica

Dengue virus (DENV) NS3 protein is a helicase that catalyzes the hydrolysis of ATP and couplesthe free energy of this reaction to the translocation on single strands and to unwind double strandedRNA. Using a fluorescent 10 base-RNA oligonucleotide (F-p-R10), we performed spectroscopictitration experiments between NS3 and RNA in the presence of different concentrations of monovalentcations at different temperatures. From the temperature dependence of the observed equilibriumassociation constant, we obtained the enthalpic and entropic contributions to the Gibbs free energychange due to the salt effect on the equilibrium between NS3 and ssRNA. Our results indicate that theassociation of NS3 to F-p-R10 is enthalpically driven under all experimental conditions tested and theeffect of salt concentration on ΔrGºobs, resides almost entirely in its entropic term T. ΔrSºobs, whichdecreased linearly with log[K+].On the other hand, we studied the effect of various ligands participating in the catalytic cicle of ATPhydrolysis on the NS3-RNA interaction. Isothermal titration calorimetry was used to determined the ofcharacteristic thermodyamic parameters of the binding between NS3h and RNA in presence of ADP, yphosphate. Our results indicate that the observed association constant for the binding between NS3hand p-R10 (the countrapart unlabel of F-p-R10) is lower in the presence than in the absence of ADP;deceasing ~3 orders of magnitude. Additionaly, in the presence of phosphate the observed associationconstant also decreases although to a lesser extent than when ADP is present.