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

LA CABABIE; JJ INCICCO; RM.GONZÁLEZ LEBRERO; LG GEBHARD; AV GAMARNIK; SB. KAUFMAN

San Moguel de tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics. XLV Reunion Anual SAB 2016.; 2016

Sociedad Argenyina de Biofísica

P { margin-bottom: 0.21cm; }Dengue virus (DENV) NS3protein is a helicase that catalyzes the hydrolysis of ATP andcouples the free energy of this reaction to the translocation onsingle strands and to unwind double stranded RNA. We have previouslypresented evidence that the observed equilibrium binding constant,which governs the interaction between DENV NS3 and ssRNA, decreaseswith increasing salt concentration (salt effect). Additionally, wedemonstrated that the formation of NS3-ssRNA complex is predominantlydriven by the favorable free energy change from the release ofcations from RNA ( 5-7 monovalent or 3 divalent cations) as a resultof the establishment of 10 ionic interactions between protein andssRNA. In this work, we studiedthe effect of pH and temperature on the interaction between NS3 andssRNA. Using a fluorescent 10 base-RNA oligonucleotide (F-p-R10), weperformed spectroscopic titration experiments NS3 / RNA in thepresence of different concentrations of mono and divalent cations atdifferent pH and temperatures. We show that pH exerts only a minorand monotonous negative effect on the observed binding constant,which is more pronounced for the unlabeled than for the 5?-labeledRNA. We propose that almost one titratable groups in the protein isdirectly involved in the interaction with RNA (in the range of pHtested) and that the protonated state of the 5?-fluorescein moietyon the labeled RNA favors the stability of the protein-nucleic acidcomplex. Finally, from the temperature dependence of the observedbinding constant, we obtained the enthalpic and entropiccontributions to the Gibbs free energy change due to the salt effecton the equilibrium between NS3 and ssRNA. Our results indicate thatthe association of NS3 to F-p-R10 is enthalpically driven under allexperimental conditions tested and the effect of salt concentrationon ∆r G0 ,resides almost entirely in its entropic term T·∆r S0, which decreased linearly with log[K+].