IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Thermodynamics of the interaction between dengue virus NS3 helicase and single stranded RNA: pH and temperature effects
Autor/es:
LEILA A. CABABIE; LEOPOLDO G. GEBHARD; RODOLFO M. GONZÁLEZ LEBRERO; SERGIO B. KAUFMAN; J. JEREMIAS INCICCO; ANDREA V. GAMARNIK
Lugar:
San Miguel de Tucumán
Reunión:
Encuentro; XLV Reunión Anual de la Sociedad Argentina de Biofísica; 2016
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
Denguevirus (DENV)NS3protein is a helicase that catalyzes the hydrolysis of ATP andcouples the free energy of this reaction to thetranslocation on single strands and unwind double strand RNA.Wehave previouslypresentedevidencethat theobserved equilibriumbindingconstant,which governsthe interaction betweenDENVNS3 andssRNA,decreaseswith increasingsalt concentration (salt effect). Additionally, we demonstrated thatthe formation ofNS3-ssRNA complexispredominantly driven by the favorable free energy change from therelease of cations from RNA(~5-7 monovalent or ~3 divalent cations) asaresultofthe establishmentof~10ionicinteractions betweenprotein andssRNA.Inthis work, we studied the effect of pH and temperature onthe interaction between NS3 and ssRNA. Usinga fluorescent 10 base-RNA oligonucleotide (F-p-R10)weperformed spectroscopictitrationexpermientsNS3 / RNA in presence of different concentrations of monoand divalent cationsat different pH and temperatures.Weshow that pH exertsonly a minor and monotonous negative effect onthe observed binding constant, which is more pronouncedfor the unlabeled than for the 5´-labeled RNA. Wepropousethat almostone titratablegroupsin the protein isdirectly involved in the interaction with RNA(inthe range of pH tested) andthat the protonated state of the 5´-fluorescein moiety on the labeledRNA favorsthe stability of the protein-nucleic acid complex.Finally, fromthetemperature dependence of theobservedbindingconstant weobtainedtheenthalpicand entropic contributions to theGibbsfree energy changeduetothesalt effect onthe equilibrium between NS3 and ssRNA.Ourresults indicate that the association of NS3 to F-p-R10 isenthalpically driven under all experimental conditions tested and theeffect of salt concentration on ΔrGºobs,resides almost entirely in its entropic term T.ΔrSºobs,which decreased linearly with log[K+].