Flexible, symmetry-directed approach to assembling protein cages

Abstract

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C 3 -symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C 4 -symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C 4 and C 3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.coiled coils | protein design | native mass spectrometry | analytical ultracentrifugation | cryoelectron microscopy

Rational Design of Supramolecular Dynamic Protein Assemblies by Using a Micelle‐Assisted Activity‐Based Protein‐Labeling Technology

“…[14] In addition, smallangle X-ray scattering (SAXS) technique has been used to determinel ow-resolution structures. [16][17][18] Most importantly,amajority of the designso nly produced static structures;i ntegration of ad ynamic behavior has yet to be explored.I na ddition, the standard genetic method is restricted to as mall set of building blocks-twenty canonical amino acids-that are used for the design purpose. [16][17][18] Most importantly,amajority of the designso nly produced static structures;i ntegration of ad ynamic behavior has yet to be explored.I na ddition, the standard genetic method is restricted to as mall set of building blocks-twenty canonical amino acids-that are used for the design purpose.…”

Metal‐dependent assembly of a protein nano‐cage

“…Protein was expressed using standard methods as previously described . Briefly, the protein was expressed in E. coli BL21(DE3) cells grown in 2xYT medium with 100 mg/L ampicillin at 37°C.…”

Engineering Protein Self‐Assembly: A New Approach for the Design of Octahedral Cages

“…Recently,S ciore et al [15] introduced an ingenious and innovative twist to this symmetry-based approach, by employing the less-conventional fusion of C 3 symmetric trimer and C 4 symmetric tetramer proteins ( Figure 1B). Nevertheless, the necessity for rigorous geometric control demands extensive designing effort.…”

“…One oligomerization domain associates as ad imer along a C 2 axis, while the other associates as at rimer along a C 3 axis. Recently,S ciore et al [15] introduced an ingenious and innovative twist to this symmetry-based approach, by employing the less-conventional fusion of C 3 symmetric trimer and C 4 symmetric tetramer proteins ( Figure 1B). However, many competingn anostructures can potentially be formed, because several symmetry point groups display the same combination of C 2 and C 3 axes ( Figure 2).…”

“…The combined effect of both symmetry axes drives the assembly of at etrahedral cage. [15] To achieve their goal, the authors chose at rimeric esterase (PDB ID:1 ZOI). Introducing these two symmetry axes is therefore not sufficient to control homogeneous self-assembly.I na ddition, strict geometricc ontrol mustb e exertedo ver the relative orientation of the two fused components.…”