Understanding the Assembly of an Artificial Protein Nanotube

Abstract

and slow-release drug-delivery through the directed packing of small molecules into the core. [7,8] Whereas traditional, inorganic, nanotube production leads to tubes that are highly uniform and therefore challenging to functionalize, designed proteins allow modular assembly of nanostructures containing multiple regions each with specific structure and function, into easily controllable smart materials and a number of protein nanotubes have been constructed. [9][10][11] Previously we demonstrated the selfassembly of a modular protein nanotube with a high aspect ratio (Figure 1). [12] The tubes are constructed from a mutant form of trp RNA-binding attenuation protein (TRAP) from Geobacillus stearothermophilus. [13][14][15][16] This small protein 11-mer forms rings ≈8.5 nm in diameter with a central pore ≈2 nm in diameter. In further experiments, TRAP has proved to be a versatile bionano building block, [17,18] and is also capable of assembling into a spherical cage. [19,20] The TRAP ring resembles a truncated cone with one face being slightly wider than the other. We arbitrarily name the narrow face "Face A" and the wide face "Face B" (Figure 1a). Each of the 11 subunits consists of 74 amino acids, many of which can be altered without significantly affecting the overall structure or stability of the fully folded and assembled 11-mer. Using the crystal structure [21] as a guide, two TRAP (trp RNA-binding attenuation protein) is a stable, ring-shaped protein that has proven useful in the development of artificial, self-assembled biological structures including a protein nanotube that assembles from a cysteine-containing mutant. While the structure of the nanotube is known in some detail, the nature of the interactions that connect the protein rings together to form tubes is not known. Here, evidence is presented that the two faces of the ring bind together using mixed interactions: On one face cysteine side chains are linked to the same face of a corresponding ring via a dithio linker molecule while the other face does not require covalent interactions and can assemble using protein-protein interactions alone. A coherent 3D model is constructed to explain the observed results, which are ultimately due to specific structural features that modulate the types of bonding that can occur. This detailed understanding offers the prospect of engineering the TRAP nanotube to endow it with bespoke properties.

Switchable supracolloidal 3D DNA origami nanotubes mediated through fuel/antifuel reactions

“…Using protein engineering concepts, there has been relevant progress to obtain protein-based nanotubes or filaments ex vivo. [5][6][7][8][9][10][11] Even though structural engineering of such proteins has advanced considerably, it remains a challenge to de novo design protein nanoparticle building blocks capable of fibrillar assembly and with elaborate control of switching interactions. In contrast, tailor-made, synthetic building blocks with defined dimensions and controlled interaction patterns could provide an alternative.…”

Protein Nanotubes: From Bionanotech towards Medical Applications

“…Mutations V69C and E50L on the monomer place the cysteines approximately 2 nm from the center of the ring on each side, with a total of 11 cysteine resides per face (Figure 5). The mutant protein is able to assemble into nanotubes reaching up to 1 µm or more in length [16,18]. An additional mutant form L50C was optimized for ideal packing of the shorter face of the ring, termed Face A, forming a tightly packed dumbbell structure stabilized by direct disulfide bonds (Figure 5).…”

“…These dumbbell-shaped dimers are then able to form bridged disulfide bonds through C69 on their wide interface (Face B) when a double-ended dithio linker such as dithiothreitol (DTT) is in solution under oxidizing conditions. This enables the assembly of the dimers into a polymeric nanotube that have higher resistance to dissociation from dilution [18].…”

“…In the original description of the TRAP PNTs [16], residues L50 and C69 allow for a hydrophobic-mediated interaction of the narrow “A” faces, and a dithio-mediated (such as via dithiothreitol, DTT) interaction of the “B” faces due to the steric bulk surrounding C69. ( b ) S Single particle analysis of the initial PNT forming “Tube TRAP” (TT) (scale bar represents 2 nm) [16], which was further modified to generate longer, more stable PNTs [18]. ( c ) Mutation L50C generates a di-cysteine mutant (TT CC ) resulting in a much more stable PNT.…”

“…Another natural system of interest has been the adaptation of viral coat proteins for the production of nanowires and targeted drug delivery. The artificial modification of multimer ring proteins such as wild-type trp tRNA-binding attenuating protein (TRAP) [16,17,18], P. aeruginosa Hcp1 [19], stable protein 1 (SP1) [20], and the propanediol-utilization microcompartment shell protein PduA [21], have successfully produced nanotubes with modified dimensions and desired chemical properties. We discuss recent advances made in using protein nanofibers and self-assembling PNTs for a variety of applications.…”