Papers by Sergio Benjamín Kaufman
<p>Initial rates of ATP hydrolysis were obtained at different concentrations of poly(A) (a)... more <p>Initial rates of ATP hydrolysis were obtained at different concentrations of poly(A) (a) or poly(C) (b). NS3h and RNA were pre-incubated for about 30 to 40 minutes in the reaction media prior to the addition of ATP. Final concentrations of RNA bases are indicated in the plots and final [NS3h] was 10 nM in all experimentsis. Continuous lines are the best fitting hyperbolas for each RNA concentration, with <i>k<sub>cat</sub></i> and <i>K<sub>M</sub></i> values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g004" target="_blank">Figure 4</a>.</p
<p><i>k<sub>cat</sub></i>, <i>K<sub>M</sub></i... more <p><i>k<sub>cat</sub></i>, <i>K<sub>M</sub></i> and <i>k<sub>cat</sub></i>/<i>K<sub>M</sub></i> values (SE) are for the substrate curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g001" target="_blank">Figure 1</a>. These parameters corresponds to a single hyperbolic function.</p
Nucleic Acids Research
The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA... more The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA replication and other stages in the flavivirus life cycle. NS3h uses energy from ATP hydrolysis to translocate along single stranded nucleic acid and to unwind double stranded RNA. Here we present a detailed mechanistic analysis of the product release stage in the catalytic cycle of the dengue virus (DENV) NS3h. This study is based on a combined experimental and computational approach of product-inhibition studies and free energy calculations. Our results support a model in which the catalytic cycle of ATP hydrolysis proceeds through an ordered sequential mechanism that includes a ternary complex intermediate (NS3h-Pi-ADP), which evolves releasing the first product, phosphate (Pi), and subsequently ADP. Our results indicate that in the product release stage of the DENV NS3h a novel open-loop conformation plays an important role that may be conserved in NS3 proteins of other flaviviruses...
<p><i>k<sub>cat</sub></i> (a), <i>K<sub>M</sub>&l... more <p><i>k<sub>cat</sub></i> (a), <i>K<sub>M</sub></i> (b) and <i>k<sub>cat</sub>/K<sub>M</sub></i> (c) values were obtained from substrate curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g003" target="_blank">Figure 3</a>. In the absence of RNA the values of <i>k<sub>cat</sub>, K<sub>M</sub></i> and <i>k<sub>cat</sub>/K<sub>M</sub></i> were 2.91 (0.03) s-1, 0.018 (0.001) mM and 16 (1) 10<sup>4</sup> M<sup>-1</sup>s<sup>-1</sup>, respectively. The limit saturating values of <i>k<sub>cat</sub>, K<sub>M</sub></i> and <i>k<sub>cat</sub>/K<sub>M</sub></i> were respectively 28 (1) s<sup>-1</sup>, 0.33 (0.03) mM and 8.4 (0.4) 10<sup>4</sup>M<sup>-1</sup>s<sup>-1</sup> for poly(A) and 16.6 (0.3) s<sup>-1</sup>, 0.30 (0.02) mM and 5.6 (0.2) 10<sup>4</sup>M<sup>-1</sup>s<sup>-1</sup> or poly(C). Continuous lines are the best fitting curves to a rational equation and are only intended to guide the eye. Error bars are the standard error of the parameters fitted to the experimental results by nonlinear regression analysis.</p
<p>Data showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.... more <p>Data showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g006" target="_blank">Figure 6</a> are plotted as functions of [poly(A)] (a, b) or [poly(C)] (c, d) at the indicated concentrations of ATP. Continuous lines proceed from simulations of the best fitting solution of the model presented below.</p
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2020
The Journal of Physical Chemistry B, 2017
Nucleic acids research, 2014
Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2... more 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 w...
PloS one, 2012
The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents a... more The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or n...
The Journal of Physical Chemistry B, 2012
Journal of Biological Chemistry, 2001
Journal of Biological Chemistry, 1999
Journal of Biological Chemistry, 2006
Journal of Biological Chemistry, 2008
FEBS Letters, 2009
MINT‐7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (... more MINT‐7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by molecular sieving (MI:0071)
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Papers by Sergio Benjamín Kaufman