Crystal Engineering of Self-Assembled Porous Protein Materials in Living Cells

Abstract

Crystalline porous materials have been investigated for development of important applications in molecular storage, separations, and catalysis. The potential of protein crystals is increasing as they become better understood. Protein crystals have been regarded as porous materials because they present highly ordered 3D arrangements of protein molecules with high porosity and wide range of pore sizes. However, it remains difficult to functionalize protein crystals in living cells. Here, we report that polyhedra, a natural crystalline protein assembly of polyhedrin monomer (PhM) produced in insect cells infected by cypovirus, can be engineered to extend porous networks by deleting selected amino acid residues located on the intermolecular contact region of PhM. The adsorption rates and quantities of fluorescent dyes stored within the mutant crystals are increased relative to those of the wild-type polyhedra crystal (WTPhC) under both in vitro and in vivo conditions. These results provide a strategy for designing self-assembled protein materials with applications in molecular recognition and storage of exogenous substances in living cell as well as an entry point for development of bioorthogonal chemistry and in vivo crystal structure analysis.

Hierarchical Self-Assembly of Proteins Through Rationally Designed Supramolecular Interfaces

“…Besides, "giant proteinosome" could be used to encapsulate hundreds of components for cell-free gene expression system (Huang et al, 2013), in cellular enzymatic reaction (Huang et al, 2014) and programmed cargo release (Liu et al, 2016). Uneo et al used self-assembled porous protein crystals for molecular recognition and storage of exogenous substances in living cell as well as applicable for bioorthogonal chemistry (Abe et al, 2017).…”

Designer protein assemblies with tunable phase diagrams in living cells

“…The design of mesoscale synthetic protein assemblies is becoming increasingly powerful to create new materials [37][38][39] and functions [40][41][42] . Moreover, as we are only beginning to grasp the complexity of proteome self-organization, new approaches are needed for characterizing and understanding mesoscale properties of protein self-assembly in cells [43][44][45][46][47][48] .…”

KAMO: towards automated data processing for microcrystals

“…Here, the power of clustering to achieve a better result is demonstrated using cypovirus polyhedra, a natural crystalline protein assembly of the polyhedrin monomer produced in infected insect cells (Abe et al, 2017). 184 small-wedge (5 ) data sets for a deletion mutant Á3 (PDB entry 5gqn) were collected automatically using ZOO and an EIGER X 9M detector (DECTRIS).…”

“…KAMO was originally developed for online use at the SPring-8 beamlines, but is now publicly available under an open-source license and can be used anywhere for small-wedge data sets. KAMO has already been used in several structure determinations from multiple microcrystals (Abe et al, 2017;Shihoya et al, 2017;Taniguchi et al, 2017;Lee et al, 2017;Miyauchi et al, 2017;Suno et al, 2017). In this article, the details of KAMO are described, followed by processing examples using synthetic and real-world data sets for the mercury-bound M 2 receptor and cypovirus polyhedra, whereby the merged structure-factor amplitudes can be obtained with minimal user intervention using KAMO.…”