Dataset Information

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

ABSTRACT: 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.

SUBMITTER: Cressiot B

PROVIDER: S-EPMC5963890 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Publications

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Cressiot Benjamin B Greive Sandra J SJ Si Wei W Pascoa Tomas C TC Mojtabavi Mehrnaz M Chechik Maria M Jenkins Huw T HT Lu Xueguang X Zhang Ke K Aksimentiev Aleksei A Antson Alfred A AA Wanunu Meni M

ACS nano 20171115 12

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 ...[more]

PMID: 29120602

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Project description:Using a recently developed binary bilayer system (BBS) consisting of two patches of laterally contacting bilayers, umbrella sampling molecular dynamics (MD) simulations were performed for quantitative characterization of protein-lipid interactions. The BBS is composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with an embedded model membrane protein, a gramicidin A (gA) channel. The calculated free energy difference for the transfer of a gA channel from DLPC (hydrophobic thickness ? 21.5 Å) to DMPC (hydrophobic thickness ? 25.5 Å) bilayers, ?G(DLPC ? DMPC), is -2.2 ± 0.7 kcal/mol. This value appears at odds with the traditional view that the hydrophobic length of the gA channel is ?22 Å. To understand this discrepancy, we first note that recent MD simulations by different groups have shown that lipid bilayer thickness profiles in the vicinity of a gA channel differ qualitatively from the deformation profile predicted from continuum elastic bilayer models. Our MD simulations at low and high gA:lipid molar ratios and different membrane compositions indicate that the gA channel's effective hydrophobic length is ?26 Å. Using this effective hydrophobic length, ?G(DLPC ? DMPC) determined here is in excellent agreement with predictions based on continuum elastic models (-3.0 to -2.2 kcal/mol) where the bilayer deformation energy is approximated as a harmonic function of the mismatch between the channel's effective hydrophobic length and the hydrophobic thickness of the bilayer. The free energy profile for gA in the BBS includes a barrier at the interface between the two bilayers which can be attributed to the line tension at the interface between two bilayers with different hydrophobic thicknesses. This observation implies that translation of a peptide between two different regions of a cell membrane (such as between the liquid ordered and disordered phases) may include effects of a barrier at the interface in addition to the relative free energies of the species far from the interface. The BBS allows for direct transfer free energy calculations between bilayers without a need of a reference medium, such as bulk water, and thus provides an efficient simulation protocol for the quantitative characterization of protein-lipid interactions at all-atom resolution.

Dataset Information

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Publications

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

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