Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.

GEBHARD LG; INCICCO JJ,; SMAL C.; GALLO M.; GAMARNIK A*; KAUFMAN SB*

NUCLEIC ACIDS RESEARCH

OXFORD JOURNALS

Año: 2014

Dengue virusnonstructural protein 3 (NS3) is a molecular motor protein that unwinds duplexRNA driven by the free energy derived from the hydrolysis of nucleosidetriphosphates. Here we report results from Pulse field gradient NMR (PFG-NMR)methods to determine the oligomeric state of NS3h, and analyses of interactionsof NS3h with ssRNA using a quantitative fluorescence titration technique.PFG-NMR spectroscopy was used for the measurement of diffusion coefficients.Results are given for effective hydrodynamic radii (inversely proportional tothe diffusion coefficient as shown Stokes-Einstein equation) of a series ofsolutions of different concentrations -20 to 250 µM- of NS3h, in the presenceand absence of short ssRNA showing that NS3h is a monomer protein in solutionin all experimental conditions tested. The stoichiometry of the NS3h-ssRNAcomplex has been examined in binding experiments with a series offluorescein-labeled ssRNA oligomers. Both the binding and the occludedsite-size of the NS3h-ssRNA complexes were 10 ± 1 residues per protein. Experimental results were well described by astatistical thermodynamic model that takes into account the overlap ofpotential binding sites and cooperative interactions between adjacent NS3molecules. Global fitting of this model to all titration curves gave a bindingsite size of 10 ± 1 bases per NS3h; an intrinsic association constant of 0.0048± 0.0001 nM-1 and a cooperativity factor ω of 3.4 ± 0.7.