Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: Structural and functional studies of Ryegrass mottle virus-encoded proteins
Project No.: 1.1.1.2/VIAA/3/19/462
Period: 36 months (1 March 2020 – 28 February 2023)
Project costs: 133 304.57 EUR
Project implementer: Dr. biol. Ina Baļķe
The aim of the project is to use Ryegrass mottle virus (RGMoV, Sobemovirus) as a model for identification of virus encoded protein structural and functional properties to better understand there roll during virus life cycle. Sobemovirus genomes are relatively well characterized, but their encoded protein structures and, in particular their role and interactions in the infection process remain to be cleared up. For virus protein 3D structure determination corresponding protein coding sequences will be cloned in to E.coli expression system vectors and overexpressed, purified and used for X-ray crystallography or Crio-EM, KMR if needed. For protein functional study localization and interaction experiments in protoplasts will be performed for proteins with fluorescent tags (whole protein or split). Fluorescent and confocal microscopy will be used as visualization methods. This information will spread light upon virus protein roll in virus life cycle and reveal crucial steps in it. That can be used for virus resistant plant development and used for other virus (mammalian) research. Also important information about potential use of virus vector for recombinant protein expression in plants will be inquired.
Information published 02.03.2020.
Project progress
1 March 2020 – 31 May 2020
Within Activity No.1, the previously created plasmids of RGMoV 3C serine protease catalytic center mutant (Procm) with and without the transmembrane domain (TMD) were expressed in E.coli expression strain C2566. The total lysates of the cells were analyzed on an SDS-PAA gel, as well as the pellet and supernatant after cell disintegration were analyzed to verify the expression level and protein solubility. As both variants of Procm are in the soluble fraction, the preparative purification of both proteins was performed on a Ni2+ affinity chromatography column, as well as additional purification on a gel filtration column to obtain a purer material for 3D structure determination was tested. Optimization of purification conditions has been performed for Procm with TMD. Catalytically active Pro was expressed and purified for functionality studies. Work has also begun on the expression and purification of P1 protein from inclusion bodies (IB).
In Activity No.3 an additional literature analysis was performed to create plasmids for protein-protein interaction studies with split fluorescent proteins (sFP). Work has started on the design of the constructs.
Information published 31.05.2020.
Progress of the project
1 June 2020 – 31 August 2020
Within Activity No.1, a purification scheme of Procm without TMD has been developed, as well as it has been purified in a preparative amount for the crystallization condition optimization study. Work continues on the optimization of Procm with TMD purification conditions, as well as on the optimal purification method selection for P1 protein purification from IB and on the studies of catalytically active Pro functionality.
During Action No.2, work has been started on testing the conditions for Procm without TMD crystallization and co-crystallization with peptide.
In Activity No.3 work has been started on the development of plasmids for sFP protein interaction studies.
Information published 31.08.2020.
Progress of the project
1 September 2020 – 30 November 2020
Within Activity No.1, work continues on the optimization of Procm with TMD purification conditions, as well as on the optimal purification method selection for P1 protein purification from IB. P16 N-terminal domain P10 was expressed in E.coli expression strain C2566. Expression and protein solubility analysis on SDS-PAA gel was performed. Purification of P10 was performed on a Ni2+ affinity chromatography column. P16 expression was performed in E.coli expression strain C2566. Analysis of P16 expression clones on SDS-PAA gel was performed. Work on catalytically active Pro functionality continues.
During Action No.2, 3D structure Procm without TMD and without peptide was determined. Testing of P16 N-terminal domain P10 crystallization conditions has been started. Preparation of a publication on previously obtained crystallization data for catalytically active Pro with and without cofactor was started.
In Activity No.3 constructs for sFP experiments was created. Work has begun on testing the expression of sFP constructs in the E.coli system.
Information published 30.11.2020.
Progress of the project
1 December 2020 – 28 February 2021
Within Action No.1, the work was continued on the optimization of P1 refolding buffers, as well as on the optimization of Procm with TMD purification conditions. Preparative expression of P16 N-terminal domain P10 in E.coli and purification for crystallization experiments were performed. A new expression vector for catalytically active ∆117Pro with an N-terminal 6 histidine tag was created for faster Pro purification. ∆117Pro was expressed in E.coli expression strain C2566, expression, protein solubility analysis on SDS-PAA gel were performed, as well as ∆117Pro was purified on Ni2+ affinity chromatography column. Work had begun on the construction of the ORFx-encoded protein Px expression vector for E.coli system.
During Action No.2 experiments for optimization of P10 crystallization conditions have been started. Work continues on the preparation of a publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.
In Activity No.3 continues the work on the expression of sFP constructs in the E.coli system. An analysis of the literature on available protoplast isolation and transfection methods has been performed.
Information published 26.02.2021.
Progress of the project
1 March 2021 – 31 May 2021
Within Action No.1, the interaction of P1 with RGMoV CP mRNA after refolding with and without Zn2+ was tested using the gel-shift method. The P1 refolding method has been developed. Using this method, P1 was purified in a preparative amount for crystallization optimization experiments. Expression of ORFx-encoded protein Px in E. coli expression strain C2566 was performed. Its expression, solubility was analyzed in SDS-PAA gel, as well as purification of Px on Ni2+ affinity chromatography column was tested. Additional purification experiments of ∆117Pro with N-terminal 6 histidine tag were performed using gel filtration.
During Action No.2 experiments for optimization of P1 crystallization conditions have been started. The experiment of optimizing P10 crystallization conditions continues. Work continues on the preparation of a publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.
In Activity No.3 has started work on compilation of protoplast isolation and transfection methods described in the literature.
Within Activity No.5, a virtual poster “Application of fluorescent proteins in plant virus research” was created within the framework of “European Scientists Night” (April 30, 2021).
Information published 31.05.2021.
Progress of the project
1 June 2021 – 31 Augusts 2021
Within Action No.1, work has been started on the optimization of Px purification of the ORFx encoded protein. Work has begun on the expression of VPg-RdRp fusion protein in E. coli expression strain C2566 and purification on a Ni2 + affinity chromatography column.
During Action No.2 the initiated optimization experiments of P1 and P10 crystallization conditions were continued. Work continues on the improvement of the publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.
In Activity No.3 continues the work on the summarization of protoplast isolation and transfection methods described in the literature.
Information published 31.08.2021.
Progress of the project
1 September 2021 – 30 November 2021
Within Action No.1, the work was continued on the optimization of the Px purification of the ORFx-encoded protein, as well as purification of VPg-RdRp fusion protein. P1 and P10 was purified in preparative amounts for co-crystallization experiments. ∆117Pro-N6H, Pro and VPg were expression and purification of in preparative amounts for FRET experiment.
During Action No.2 experiments with P1 and P10 crystallization were continued. Co-crystallization experiments with P1 and P10 have been started. Work continues on editing of the publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.
In Activity No.3 protoplasts were isolated and transfected with a control plasmid. The transfected protoplasts were analyzed with a confocal microscope.
Information published 30.11.2021.
Progress of the project
1 December 2021 – 28 February 2022
Within Action No.1, ∆117Pro-N6H, ∆117Pro and VPg FRET experiments were performed at different temperatures and in different buffers, but no changes in fluorescence level were observed, which would indicate cleavage of the peptide. Conditions should be optimized or the length of the peptide reduced. ∆50Procm was expressed and purified in preparative quantities, it will be used as a substrate for analysis of ∆117Pro in cis cleavage dynamics with and without cofactor – VPg.
During Action No.2 the work on crystallization experiments with P1 and P10 was continued. The manuscript on the previously obtained crystallization data for the catalytically active Pro with and without cofactor was supplemented with data on the crystal structure of ∆117Procm, and the manuscript was edited.
In Activity No.3 work had started on isolation and transfection of oat protoplasts with the developed plasmid constructs for protein interaction studies.
Information published 28.02.2022.
Progress of the project
1 March 2022 – 31 May 2022
Within Action No.1 an analysis of ∆117Pro in cis cleavage dynamics with and without cofactor – VPg was performed ∆50Procm was used as a substrate. ∆117Pro in cis cleavage dynamics was tested in three temperatures – +4°C, +23°C, +37°C, and without or with 2 mM, 5 mM CaCl2, 2 mM MgCl2 and 2mM ZnCl2.
During Action No.2 the work on crystallization experiments with P1 and P10 was continued. The manuscript on the previously obtained crystallization data, for the catalytically active Pro with and without cofactor was supplemented with data from ∆117Pro in cis cleavage dynamics experiment.
In Activity No.3 work continued on the isolation and transfection of oat protoplasts with the developed plasmid constructs for protein interaction studies.
Information published 31.05.2022.
Progress of the project
1 June 2022 – 31 August 2022
Within Action No.1 was performed the expression and purification of ∆50Pro-C6H in preparative amounts, which will be used for analysis of in trans cleavage with and without the cofactor – VPg. In addition, P1 optimization experiments were performed to improve solubility by expressing P1 in BL21(DE3) E. coli expression strain according to the expression and P1 purification conditions published by Gillet et al., 2013.
During Action No.2 the work on co-crystallization and crystallization experiments with P1 and P10 was continued. The manuscript on previously obtained crystallization data for catalytically active ∆117Pro with and without cofactor was supplemented with ∆50Pro-C6H in trans cleavage dynamics data.
In Activity No.3 continues work on transfection of oat protoplasts with the created plasmid constructs, for studies of protein interactions. An analysis of the dynamics of ∆117Pro in cis cleavage with and without the cofactor – VPg was performed, using ∆50Procm as a substrate without and with 100 mM NaCl. Analyzes of ∆50Pro-C6H in trans cleavage with and without the cofactor – VPg, as well as without and with 2 mM, CaCl2, 2 mM MgCl2, 2mM ZnCl2 and 100 mM NaCl were carried out.
Information published 31.08.2022.
Progress of the project
1 September 2022 – 30 November 2022
Within Activity No.1 was performed expression of VPg-RdRp and P16 in C2566 E. coli expression strain and purification for crystallization and co-crystallization experiments. Initiated co-expression of RGMoV-encoded proteins in an E. coli expression system to identify potential protein-protein interactions.
During Activity No.2 a publication manuscript on crystallization data on catalytically active ∆117Pro, with and without cofactor and catalytically inactive ∆117Pro was submitted for publication in PNAS. In silico 3D structure modelling for P16, Px and RdRp using AlphaFold was performed, as well as the identification of structural elements with PsiPred.
In Activity No.3 continues work on the transfection of oat protoplasts with the created plasmid constructs for protein interaction and localization studies.
Mobility: Completed 3-month mobility (01.07.2022 – 30.09.2022) at Tallinn Technical University.
Information published 30.11.2022.
Progress of the project
1 December 2022 – 28 February 2023
Within Activity No.1 initiated optimization of VPg-RdRp and P16 expression and purification for crystallization and co-crystallization experiments. Continued co-expression of RGMoV-encoded proteins in an E. coli expression system to identify potential protein-protein interactions.
During Activity No.2 a publication manuscript on crystallization data on catalytically active ∆117Pro, with and without cofactor and catalytically inactive ∆117Pro was reworked and submitted for publication in the journal IJMS.
In Activity No.3 continues work on the transfection of oat protoplasts with the created plasmid constructs for protein interaction and localization studies. Results from a bacterial two-hybrid system on RGMoV protein-protein and potential protein-nucleic acid interactions were summarized.
Information published 28.02.2023.