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1.D.252.  The Sculpted Ion Conducting Beta Barrel-NanoPore (SIC-BB-NP) Family 

Transmembrane β-barrels have potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Berhanu et al. 2024 described a general approach to designing transmembrane β-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameters, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.

If the nanopore has 8 β-strands, it has an inner diameter of 16.4 Å; if 10 β-strands, it has an inner diameter of 19.4 Å; if it has 10 β-strands, it has an inner diameter of 22.8 Å; and  if it has 14 β-strands, it has an inner diameter of 26.4 Å (Berhanu et al. 2024).  The experimentally determined nanopore structures closely align with the computationally designed models.  These and other features such as the sizes and shapes of these protein porins determine their conductivities. Whereas the shapes of globular proteins are largely determined by the packing of hydrophobic residues in a central core, the BB-NP shapes can be specified by strategic placement of glycine residues at which bending takes place to reduce strain. As previously observed for eight-stranded BB-NPs, a delicate balance between the optimization of tertiary structure energy and negative design (introduction of locally frustrated residues) to disfavor premature β-strand formation before membrane insertion was critical for the expression of the larger BB-NPs in E. coli inclusion bodies (Berhanu et al. 2024).

The monomeric BB-NP design—similar to that used by the naturally occurring nanopores used for sensing applications — can be solubilized in detergent and spontaneously inserted into a planar lipid membrane following dilution. The most stable nanopores allowed up to 2 hours of quiet recording, thanks to the use of short loops compatible with TMB folding to connect the β-strands (Berhanu et al. 2024).

References associated with 1.D.252 family:

Berhanu, S., S. Majumder, T. Müntener, J. Whitehouse, C. Berner, A.K. Bera, A. Kang, B. Liang, N. Khan, B. Sankaran, L.K. Tamm, D.J. Brockwell, S. Hiller, S.E. Radford, D. Baker, and A.A. Vorobieva. (2024). Sculpting conducting nanopore size and shape through de novo protein design. Science 385: 282-288. 39024453