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Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.
- Source :
-
Viruses [Viruses] 2020 Dec 23; Vol. 13 (1). Date of Electronic Publication: 2020 Dec 23. - Publication Year :
- 2020
-
Abstract
- Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.
- Subjects :
- Adenosine Triphosphatases
Bacteriophages ultrastructure
Cloning, Molecular
Gene Expression
Models, Molecular
Recombinant Proteins
Spectrum Analysis
Structure-Activity Relationship
Struvite
Viral Proteins chemistry
Viral Proteins genetics
Viral Proteins metabolism
Virion ultrastructure
Virus Assembly
Bacteriophages isolation & purification
Bacteriophages physiology
Chemical Phenomena
DNA Packaging
DNA, Viral
Lactococcus lactis virology
Subjects
Details
- Language :
- English
- ISSN :
- 1999-4915
- Volume :
- 13
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Viruses
- Publication Type :
- Academic Journal
- Accession number :
- 33374840
- Full Text :
- https://doi.org/10.3390/v13010015