Shape‐Anisotropic Assembly of Protein Nanocages with Identical Building Blocks by Designed Intermolecular π–π Interactions

Abstract Protein lattices that shift the structure and shape anisotropy in response to environmental cues are closely coupled to potential functionality. However, to design and construct shape‐anisotropic protein arrays from the same building blocks in response to different external stimuli remains challenging. Here, by a combination of the multiple, symmetric interaction sites on the outer surface of protein nanocages and the tunable features of phenylalanine‐phenylalanine interactions, a protein engineering approach is reported to construct a variety of superstructures with shape anisotropy, including 3D cubic, 2D hexagonal layered, and 1D rod‐like crystalline protein nanocage arrays by using one single protein building block. Notably, the assembly of these crystalline protein arrays is reversible, which can be tuned by external stimuli (pH and ionic strength). The anisotropic morphologies of the fabricated macroscopic crystals can be correlated with the Å‐to‐nm scale protein arrangement details by crystallographic elucidation. These results enhance the understanding of the freedom offered by an object's symmetry and inter‐object π−π stacking interactions for protein building blocks to assemble into direction‐ and shape‐anisotropic biomaterials.


Table of Contents
800 mM for 2D structure while at the same time neutralizing the system.The energy of the systems was minimized via the steepest descent algorithm.Afterwards, the systems were equilibrated, first in the NVT ensemble (i.e., with a constant number of molecules, volume, and temperature) for 3 ns and second for 1 ns in the NpT ensemble at 293 K and 1.0 bar.Their interaction energy [6]   =   +   consisting of Coulomb and Lennard-Jones (LJ) contributions was determined, which was accomplished by rerunning the simulation using gmx mdrun -rerun to obtain the energies, which were processed using gmx energy to calculate ECoul and ELJ between the 3FF monomers.

Statistical Analysis
All data were analyzed and plotted using Origin 2019.The data of zeta potential of 3FF were presented as means ± standard deviation and analyzed with a sample size of n = 3.The results in Figure S10 were analyzed using a one-way ANOVA with Tukey's post-hoc test.The significance values are indicated with **** as p < 0.0001.[b] CC1/2 is the correlation coefficient of the half datasets.
[c] Rwork = ∑hkl | |Fobs| -|Fcalc| | / ∑hkl |Fobs|, where Fobs and Fcalc is the observed and the calculated structure factor, respectively.Rfree is the crossvalidation R factor for the test set of reflections (5% of the total) omitted in model refinement.

Figure S3 .
Figure S3.The outer surfaces related to the C2, C3, and C4 axes show different curvatures.The outer surface around the C3 has the largest curvature.

Figure S4 .
Figure S4.Location and orientation of D123 in wild-type HuHF.D123 residues are nearby the C3 rotation axes, which are highlighted in red.The center of the C3 pore is represented by blue sphere.

Figure S10 .
Figure S10.(a) Zeta potential of 3FF at different pH (means ± standard deviation, n = 3, ****p < 0.0001).Results were analyzed using a one-way ANOVA with Tukey's post-hoc test.(b) Effect of pH on the surface electrostatics of 3FF.Poisson−Boltzmann electrostatic potential mapped onto molecular surfaces of a monomer and the C3 surfaces.The surface electrostatics (electrostatic potential expressed in the units of ± 5 kBT/e) is generated using the APBS tool, where red, white, and blue patches indicate the presence of negatively, neutrally, and positively charged amino acid residues, respectively.

Figure S12 .
Figure S12.Comparison of interaction energy (Eint) consisting of Coulomb and Lennard-Jones (LJ) contributions, separation distances and interfacial contact areas of interacted C3 interfaces (joint A to F) in 1D lattice.

Figure S13 .
Figure S13.Closeup views of the intermolecular interactions at joint B-F in 1D lattice.

Figure S14 .
Figure S14.Comparison of interaction energy (Eint) consisting of Coulomb and Lennard-Jones (LJ) contributions, separation distances and interfacial contact areas of interacted C3 interfaces 2D lattice.
[a] Highest resolution shell is shown in parentheses.