Bottom-up design of Ca²⁺ channels from defined selectivity filter geometry

Area of Science:

• Biophysics
• Protein Engineering
• Computational Biology

Background:

• Native ion channels are vital in biology and engineered for tools.
• Designing precise ion selectivity filters has been a major challenge.
• Existing methods lack atomic-level precision for metal-coordinating residues.

Purpose of the Study:

• To develop a bottom-up computational approach for designing Ca2+ channels.
• To create channels with tunable selectivity filter geometries and coordination numbers.
• To validate the designed channels' function and structural accuracy.

Main Methods:

• Utilized RFdiffusion, a computational protein design method.
• Designed symmetric oligomeric channels with varying Ca2+ selectivity filter geometries.
• Employed patch-clamp experiments and cryogenic electron microscopy for validation.

Main Results:

• Successfully designed and assembled homogeneous pore-containing protein particles.
• Demonstrated higher Ca2+ conductance compared to other ions (Na+, Sr2+, Mg2+).
• Cryo-EM confirmed high accuracy, with designed structures closely matching models.

Conclusions:

• The bottom-up design approach enables precise construction of selective ion channels.
• This method facilitates testing structure-selectivity hypotheses.
• Provides a roadmap for creating custom ion channels for diverse applications.