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Computational design of transmembrane pores.

ABSTRACT: Transmembrane channels and pores have key roles in fundamental biological processes¹ and in biotechnological applications such as DNA nanopore sequencing^2-4, resulting in considerable interest in the design of pore-containing proteins. 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 that are capable of conducting ions selectively or are large enough to enable the 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 ?-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy 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 channels and pores for a wide variety of applications.

SUBMITTER: Xu C

PROVIDER: S-EPMC7483984 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Computational design of transmembrane pores.

Xu Chunfu C Lu Peilong P Gamal El-Din Tamer M TM Pei Xue Y XY Johnson Matthew C MC Uyeda Atsuko A Bick Matthew J MJ Xu Qi Q Jiang Daohua D Bai Hua H Reggiano Gabriella G Hsia Yang Y Brunette T J TJ Dou Jiayi J Ma Dan D Lynch Eric M EM Boyken Scott E SE Huang Po-Ssu PS Stewart Lance L DiMaio Frank F Kollman Justin M JM Luisi Ben F BF Matsuura Tomoaki T Catterall William A WA Baker David D

Nature 20200826 7823

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,1 ...[more]

PMID: 32848250

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Project description:Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.

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Computational design of transmembrane pores.

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Computational design of transmembrane pores.

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