Setting the experimental conditions for the study of the interaction between Zika virus NS3 helicase and single-stranded RNA by spectroscopic titration

MIKKELSEN, EVELYN; INCICCO, JJ; KAUFMAN, SERGIO B.; GEBHARD, LG; ROSSI, RC; CABABIE, LA; GAMARNIK, AV

Rosario, Santa Fe

Congreso; L Reunión Anual SAB; 2022

Sociedad Argentina de Biofísica

Zika virus non-structural protein 3 (NS3) is a multifunctional protein with two functional domains: an N-terminal domain with protease activity and a C-terminal domain which presents RNA helicase, nucleoside 5? phosphatase (NTPase) and 5?-terminal RNA triphosphatase (RTPase) activities. RNA helicases are motor proteins that catalyze the hydrolysis of nucleoside triphosphates (NTPs) which provides the driving force for the rearrangement of the RNA structures. RNA helicases participate in virtually all processes of RNA metabolism.NS3 functions depend on its ability to interact with RNA. Studying the properties of this interaction, such as stoichiometry, apparent and intrinsic association constants, binding site size, cooperativity, etc is necessary to understand functional aspects of this enzyme and to propose models of the mechanism of enzyme functioning, including translocation on RNA and catalysis of RNA unwinding.In this work we study the binding of NS3 helicase to single-stranded RNA (ssRNA) by quantitative fluorescence titrations using a fluorescein labeled ssRNA oligonucleotide 10 nucleotides in length. We present preliminary titration curves as well as assays carried out in order to optimize experimental conditions. To djust the titration medium we evaluated the effect of different variables, such as the concentrations of KCl and CHAPS detergent and pH, on the protein stability in solution and on the fluorescent signal of the NS3 helicase-ssRNA complex.