TAILIEUCHUNG - Báo cáo khoa học: Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein

Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPDN5) inEscherichia coliand showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. | iFEBS Journal Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein Marie-Hélène Tremblay1 Nathalie Majeau1 Marie-Eve Laliberte Gagne1 Katia Lecours2 Helene Morin1 Jean-Baptiste Duvignaud1 Marilene Bolduc1 Nicolas Chouinard1 Christine Pare1 Stephane Gagne2 and Denis Leclerc1 1 Centre de Recherche en Infectiologie Univeriste Laval Quebec Canada 2 Departement de Biochimie Universite Laval Quebec Canada Keywords assembly coat protein nucleocapsid papaya mosaic virus potexvirus Correspondence Denis Leclerc Professeur adjoint Centre de Recherche en Infectiologie Pav. CHUL Universite Laval 2705 boul. Laurier Quebec PQ G1V 4G2 Canada Tel 1418 654 2705 ext. 47517 Fax 1418 525 4444 ext. 42026 E-mail Received 20 July 2005 revised 29 September 2005 accepted 25 October 2005 doi Papaya mosaic potexvirus PapMV coat protein CP was expressed CPDN5 in Escherichia coli and showed to self assemble into nucleocapsid like particles NLPs . Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80 was found as a multimer of 450 kDa 20 subunits arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer 150 nm than the recombinant CPDN5 and 100 percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPDN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV. Papaya mosaic potexvirus PapMV is a member of the potexvirus

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