Computational design of transmembrane pores

Area of Science:

• Biophysics
• Structural Biology
• Biotechnology

Background:

• Transmembrane channels and pores are crucial for biological processes and applications like DNA nanopore sequencing.
• Designing stable, well-defined transmembrane protein pores for selective ion conduction or molecule passage is a significant challenge.
• Previous work includes synthetic peptides forming channels and advances in de novo membrane protein design.

Purpose of the Study:

• To computationally design stable, monodisperse protein pores formed by concentric alpha-helix rings.
• To structurally and functionally characterize these designed pores in both water-soluble and transmembrane forms.
• To demonstrate the potential of these designer pores for applications in ion selectivity and molecule transport.

Main Methods:

• Computational protein design of concentric alpha-helix ring structures.
• X-ray crystallography to determine the structure of water-soluble pore forms.
• Patch-clamp electrophysiology to assess ion transport in cell membranes.
• In vitro protein synthesis and liposome incorporation for molecule passage studies.
• Cryo-electron microscopy to visualize the transmembrane pore structure.

Main Results:

• Crystal structures of designed 12- and 16-helix pores closely matched computational models.
• Transmembrane 12-helix pores exhibited selective potassium over sodium ion passage in insect cells.
• Transmembrane 16-helix pores facilitated the passage of a small-molecule fluorophore (biotinylated Alexa Fluor 488) in liposomes.
• Cryo-electron microscopy confirmed the structure of the 16-helix transmembrane pore.

Conclusions:

• The study successfully designed and validated stable, well-defined transmembrane protein pores.
• These designer pores demonstrate selective ion conduction and molecule transport capabilities.
• The findings open avenues for creating custom protein channels and pores for diverse biotechnological applications.