Benjamin Cressiot, Sandra J. Greive, Wei Si, Tomas C. Pascoa, Mehrnaz
Mojtabavi, Maria Chechik, Huw T. Jenkins, Xueguang Lu, Ke Zhang, Aleksei
Aksimentiev, Alfred A. Antson, and Meni Wanunu.
Porphyrin-assisted docking of a thermophage portal protein into lipid
bilayers: Nanopore engineering and characterization.
ACS Nano, 11:11931-11945, 2017.
(PMC: PMC5963890)
CRES2017-AA
Nanopore-based sensors for nucleic acid sequencing and single-molecule
detection typically employ pore-forming membrane proteins with hydrophobic
external surfaces, suitable for insertion into a lipid bilayer. In contrast,
hydrophilic pore-containing molecules, such as DNA origami, have been
shown to require chemical modification to favor insertion into a lipid
environment. In this work, we describe a strategy for inserting polar proteins
with an inner pore into lipid membranes, focusing here on a circular 12-subunit
assembly of the thermophage G20c portal protein. X-ray crystallography,
electron microscopy, molecular dynamics, and thermal/chaotrope
denaturation experiments all find the G20c portal protein to have a highly
stable structure, favorable for nanopore sensing applications. Porphyrin
conjugation to a cysteine mutant in the protein facilitates the protein’s
insertion into lipid bilayers, allowing us to probe ion transport through the
pore. Finally, we probed the portal interior size and shape using a series of
cyclodextrins of varying sizes, revealing asymmetric transport that possibly
originates from the portal’s DNA-ratchet function.
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