Kinetic analysis of the interaction between dengue virus NS3 helicase and ssRNA

J. JEREMÍAS INCICCO; LEILA A. CABABIE; LEOPOLDO G. GEBHARD; ANDREA V. GAMARNIK; RODOLFO M. GONZÁLEZ-LEBRERO; SERGIO B. KAUFMAN

Steamboat, Colorado

Congreso; Helicases and Nucleic-Acid Based Machines: From Mechanism to Insight into Disease; 2015

FASEB

h3 { margin-top: 0.17in; margin-bottom: 0.04in; direction: ltr; line-height: 100%; text-align: left; orphans: 2; widows: 2; }h3.western { font-family: "Times New Roman",serif; font-size: 12pt; font-weight: normal; }h3.cjk { font-family: "Calibri"; font-size: 12pt; font-weight: normal; }h3.ctl { font-family: "Times New Roman"; font-size: 10pt; font-weight: normal; }p { margin-bottom: 0.1in; direction: ltr; line-height: 120%; text-align: left; orphans: 2; widows: 2; }p.western { font-family: "Times New Roman",serif; font-size: 12pt; }p.cjk { font-size: 12pt; }p.ctl { font-size: 12pt; } Dengue virus nonstructural protein 3 (NS3) is a molecular motor protein that unwinds duplex RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. In previous studies we examined the interaction of NS3h with ssRNA under equilibrium conditions and we found that the interaction is characterized by minimum and occluded site sizes both of 10 nucleotides. In this presentation we show results from the study of the kinetic mechanism of this interaction obtained from stopped-flow experiments. A series of 5?-fluorescein-labeled oligonucleotides of different lengths (11 to 21 nucleotides) with a repetitive sequence of five nucleotides (AGUUG) were employed and the binding time courses to the NS3h was monitored by the fluorescence increase observed upon excitation at 495 nm. Reaction were carried out at 25ºC in buffer B (MOPS 25 mM, pH 6.4, KCl 100 mM (total K+), EDTA 0.50 mM, MgCl2 2.0 mM). The binding time courses observed under pseudofirst order conditions required a minimum of three exponential terms for an adequate fit. A three-steps kinetic model was able to describe the experimental time courses and provided best-fitting estimates of the rate constants for each oligonucleotide tested.