title: Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.
abstract: Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.
authors.affiliation: Fundación Instituto Leloir-Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
authors.forename: Leopoldo G
authors.initials: LG
authors.lastname: Gebhard
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, C1113AAD, Argentina.
authors.forename: J Jeremías
authors.identifier: http://orcid.org/0000-0003-2467-4504
authors.initials: JJ
authors.lastname: Incicco
authors.affiliation: Fundación Instituto Leloir-Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
authors.forename: Clara
authors.initials: C
authors.lastname: Smal
authors.affiliation: Fundación Instituto Leloir-Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Gallo
authors.affiliation: Fundación Instituto Leloir-Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
authors.forename: Andrea V
authors.initials: AV
authors.lastname: Gamarnik
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, C1113AAD, Argentina sbkauf@qb.ffyb.uba.ar.
authors.forename: Sergio B
authors.initials: SB
authors.lastname: Kaufman
mesh_terms: D001665:Binding Sites; D003716:Dengue Virus / Q000201:enzymology*; D011061:Poly A / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D011061:Poly A; D012313:RNA; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases