Designed and biologically active protein lattices

Abstract

Versatile methods to organize proteins in space are required to enable complex biomaterials, engineered biomolecular scaffolds, cell-free biology, and hybrid nanoscale systems. Here, we demonstrate how the tailored encapsulation of proteins in DNA-based voxels can be combined with programmable assembly that directs these voxels into biologically functional protein arrays with prescribed and ordered two-dimensional (2D) and three-dimensional (3D) organizations. We apply the presented concept to ferritin, an iron storage protein, and its iron-free analog, apoferritin, in order to form single-layers, double-layers, as well as several types of 3D protein lattices. Our study demonstrates that internal voxel design and inter-voxel encoding can be effectively employed to create protein lattices with designed organization, as confirmed by in situ X-ray scattering and cryo-electron microscopy 3D imaging. The assembled protein arrays maintain structural stability and biological activity in environments relevant for protein functionality. The framework design of the arrays then allows small molecules to access the ferritins and their iron cores and convert them into apoferritin arrays through the release of iron ions. The presented study introduces a platform approach for creating bio-active protein-containing ordered nanomaterials with desired 2D and 3D organizations.

Cryo-electron tomography related radiation-damage parameters for individual-molecule 3D structure determination

“…To produce an even thinner ice film, a small amount of detergent (>0.1%) can be added into the sample solution to reduce the liquid surface tension if the detergent is compatible with the sample. Similarly, the area of the ice film should also be another function of radiation damage due to the higher heat capacity of the larger ice as described in the previous publications (shown in Figure 8 ) ( Zhang and Ren, 2012 ; Lei et al, 2018 ; Wang et al, 2021b ). Large area thin ice film can be achieved by using the lacey carbon film–coated TEM grid ( Zhang and Ren, 2012 ; Segrest et al, 2015 ).…”

“…(D) Representative 3D density maps after missing-wedge correction by LoTToR. Reproduced with permission ( Wang et al, 2021b ).…”

The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles

“…Since H6 also allows simple, one-step purification of any protein by IMAC [2,3,60,61], the dual role of the peptide shows benefits from both the bioproduction and purification side, and the nanofabrication process itself, through selfassembling. In contrast to other more refined protein-specific engineering approaches [17][18][19][20][62][63][64], the protein self-assembling process promoted by H6 tails can be universally applied to any protein of clinical interest. The data shown here indicate that the recombinant production of distinct His-tagged nanobody-GFP fusions result in at least two distinguishable conformers of the protein that assemble into two categories of NPs.…”

“…However, the data presented here also reveal a biophysical variability in the resulting materials linked to the conformational spectrum of the recombinant proteins, specific of the type of soft material that they generate. This concept, irrelevant in the use of more rigid building blocks for material design, might be highly relevant in the emerging field of protein-based materials for clinical applications, which is seen as a broad and promising technology for a diversity of therapeutic applications [6,[17][18][19][20][21]62]. Being this fact an issue with regulatory implications, the awareness of such potentially disparate assembling makes possible a proper quality control of a clinically oriented product by a simple separation of relevant peaks during the chromatographic step of the production process.…”

“…In fact, this principle sustains the formation of the secretory granules in the mammalian endocrine system [7,8] and of different types of amyloidal and non-amyloidal protein materials existing in nature [4,7,[9][10][11][12][13][14][15][16]. The simplicity of His-rich peptides used as architectonic agents at the nanoscale offers an interesting alternative to more refined approaches to control protein self-assembling, that might require more sophisticated protein engineering [17][18][19][20][21].…”