ABSTRACT Although the ϕX174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein. IMPORTANCE The H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA-translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the α-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long α-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using in-frame insertions and deletions as presented herein.
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Translocation of soft phytoglycogen nanoparticles through solid-state nanochannels
Phytoglycogen nanoparticles are soft, naturally-derived nanomaterials with a highly uniform size near 35 nm. Their interior is composed of a highly-branched polysaccharide core that contains more than 200% of its dry mass in water. In this work, we measure the translocation of phytoglycogen particles by observing blockade events they create when occluding solid-state nanochannels with diameters between 60 and 100 nm. The translocation signals are interpreted using Poisson–Nernst–Planck calculations with a “hardness parameter” that describes the extent to which solvent can penetrate through the interior of the particles. Theory and experiment were found to be in quantitative agreement, allowing us to extract physical characteristics of the particles on a per particle basis.
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- Award ID(s):
- 1651002
- NSF-PAR ID:
- 10147604
- Date Published:
- Journal Name:
- Journal of Materials Chemistry B
- Volume:
- 7
- Issue:
- 41
- ISSN:
- 2050-750X
- Page Range / eLocation ID:
- 6428 to 6437
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C9TB01048C
