Computational design of transmembrane pores

Abstract

Protein pores play key roles in fundamental biological processes 1 and biotechnological applications such as DNA nanopore sequencing 2 – 4 , and hence the design of pore-containing proteins is of considerable scientific and biotechnological interest. Synthetic amphiphilic peptides have been found to form ion channels 5 , 6 , and there have been recent advances in de novo membrane protein design 7 , 8 and in redesigning naturally occurring channel-containing proteins 9 , 10 . However, the de novo design of stable, well-defined transmembrane protein pores capable of conducting ions selectively or large enough to allow passage of small-molecule fluorophores remains an outstanding challenge 11 , 12 . Here, we report the computational design of protein pores formed by two concentric rings of ɑ-helices that are stable and mono-disperse in both water-soluble and transmembrane forms. Crystal structures of the water-soluble forms of a 12 helical and a 16 helical pore are close to the computational design models. Patch-clamp electrophysiology experiments show that the transmembrane form of the 12-helix pore expressed in insect cells allows passage of ions across the membrane with high selectivity for potassium over sodium, which is blocked by specific chemical modification at the pore entrance. The transmembrane form of the 16-helix pore, but not the 12-helix pore, allows passage of biotinylated Alexa Fluor 488 when incorporated into liposomes using in vitro protein synthesis. A cryo-EM structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer pores for a wide variety of applications.

Molecular Simulations of Hydrophobic Gating of Pentameric Ligand Gated Ion Channels: Insights into Water and Ions

“… 74 Importantly, this analysis demonstrates that to a first approximation the behavior of a hydrophobic gate can be described well by just the local pore radius profile and hydrophobicity, which enables robust prediction of the functional state of new channel structures and also provides a clear design principle for hydrophobic gates in synthetic nanopores. 76 …”

Prospects of Observing Ionic Coulomb Blockade in Artificial Ion Confinements

“…That would allow us to achieve a synthetic model of a cell’s ion channels on a discrete channel level and benefit from step-by-step additions to a solid-state platform’s controls. As a result, we may expect to obtain crucial insights on gating, strain, temperature control, etc., to use as inputs in the bottom-up computation design of biopores [ 82 ], which have already proven themselves as a unique platform for sensing and single-molecule experiments [ 83 ]. So far, no further ICB signatures have been experimentally demonstrated as conductance oscillations and Coulomb staircase, even though these have been predicted for biological nanopores [ 17 ].…”

Biological Nanopores: Engineering on Demand

“…Compared to water-soluble proteins, there are significantly fewer reports of the synthesis of de novo designed membrane proteins [220]. However, significant progress has been made in both the de novo design [121,221] and the adaptation of existing natural structures [120]. Synthetic nanopores composed of lipid-modified DNA may also provide crucial advances toward the detection of challenging molecules [222].…”

“…Some wt α-helical pores spontaneously assemble into oligomers with a varying number of subunits, which increases the range of targets that can be detected with these pores [119]. In this respect, pores containing an α-helical transmembrane region may be preferred candidates for de novo design [120,121].…”