Expression, purification and structural characterization of capsidial proteins coded by grapevine virus a and grapevine virus b

• Grant number: 11/23524-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: April 02, 2012
• End date: July 01, 2012
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Raghuvir Krishnaswamy Arni
• Grantee: Vinícius dos Santos Santana
• Supervisor: Christian Betzel
• Host Institution: Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP). Campus de São José do Rio Preto. São José do Rio Preto , SP, Brazil
• Institution abroad: Universität Hamburg (UHH), Germany
• Associated to the scholarship: 10/00896-6 - Expression, purification and structural characterization of capsidial proteins coded by grapevine virus a and grapevine virus b, BP.DR

Abstract

Grapevine virus A and B (GVA and GVB) are single stranded RNA viruses, responsible for diseases in grapevines and are considered to be of great economic importance for viticulture worldwide. This project aims to understand on the atomic level the proteins considered to be responsible for virus cell-to-cell movement, the mechanism of recognition by cellular components of the vector and its host, and the mechanism of spontaneous capsid formation ("self-assembly"). Thus, the structural data will be important to understand the specific interactions that may serve as a target for future capsid disassembly and virus transmission. Grapevine virus B coat protein was expressed but it was found exclusively in inclusion bodies. Several protocols have been thoroughly tested to obtain soluble proteins. Using the protocol described by Studier (2005), soluble and properly structured protein was obtained. Circular Dichroism experiments were conducted to ascertain the degree of folding and secondary structure content of the protein. Deconvolution data indicated 71% ±-helix, 5% ²-sheet and 24% other structures. We still faced problems during the concentration of the protein that showed a strong tendency to precipitate after 24 hours, but buffers screening resulted in a significant improvement of protein stability and solubility (concentration above 7 mg / mL). Dynamic light scattering experiment indicated a high degree of aggregation- possible capsid formation (hydrodynamic radius of 100nm). With this collaboration, we intend to obtain the proteins in native state, monomeric and monodisperse, to ensure a complete structural characterization using dynamic light scattering, nuclear magnetic resonance, small angle X-ray scattering and crystallography techniques. In addition, we will learn new techniques that we intend to apply to our laboratory in the future. (AU)