Design of an In‐Cell Protein Crystal for the Environmentally Responsive Construction of a Supramolecular Filament

Abstract

Protein assemblies can be designed for development of nano–bio materials. This has been achieved by modulating protein–protein interactions. However, fabrication of highly ordered protein assemblies remains challenging. Protein crystals, which have highly ordered arrangements of protein molecules, provide useful source matrices for synthesizing artificial protein assemblies. Here, we describe construction of a supramolecular filament structure by engineering covalent and non‐covalent interactions in a protein crystal. Performing in‐cell crystallization of Trypanosoma brucei cysteine protease cathepsin B (TbCatB), we achieved a precise arrangement of protein molecules while suppressing random aggregation due to disulfide bonds. We succeeded in synthesizing bundled filament from the crystals by autoxidation of cysteinyl thiols after the isolation of the crystals from living cells.

Segregated Protein–Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons

“…Protein‐based crystalline materials are advancing in sophistication,[ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] including in vivo construction[ 6 , 7 , 11 , 13 ] and industrial application,[ 11 , 13 ] although reliable self‐assembling systems are scarce. Tried‐and‐tested systems such as ferritin [8] and polyhedrin[ 7 , 11 ] are the main focus while ligand‐mediated assembly continues to deliver remarkable bionanoarchitectures with the potential for customization.…”

“…Few examples of engineered protein‐based materials with comparable structural complexity have been reported. [ 3 , 4 , 5 , 6 ] Here, we describe the manufacture and structure elucidation of segregated crystalline assemblies that contain layers of low and high porosity in which the protein material is rigid or dynamic, respectively.…”

Protein Assembly by Design

“…Abe et al later used a similar approach to isolate covalently linked protein filaments from Trypanosoma brucei cysteine protease cathepsin B (TbCatB) crystals, formed by overexpression of R92C/T223C TbCatB in baculovirus-infected insect cells upon oxidation (Figure 44a). 416 Monomers form 1D arrays within the P4 2 2 1 2 crystals of TbCatB assembled in vivo. The mutant R92C/T223C was selected because the 5.9 Å Cα−Cα distance for the residues in the wildtype crystal was deemed appropriate for disulfide bond formation by oxidation (Figure 44b).…”

“…However, rational construction of 1D assemblies, which involves control over length, width, and assembly dynamics, remains a significant challenge. Extended 1D assemblies encompass several common morphologies, from flexible filaments and nanowires ,,− to nanotube structures. ,,, There are also several subtypes of nanotubes, as they can be composed of stacked protein rings, , assembled through the association of helical filaments or formed when nascent 2D sheets roll up into a hollow structure due to inherent curvature or kinetic effects. , Potential applications for extended 1D assemblies vary with morphology. They span broad functions across different length scales, from scaffolding and encapsulation of functional moieties at the nanoscale to bulk material such as gelation.…”

“…Abe et al later used a similar approach to isolate covalently linked protein filaments from Trypanosoma brucei cysteine protease cathepsin B (TbCatB) crystals, formed by overexpression of R92C/T223C TbCatB in baculovirus-infected insect cells upon oxidation (Figure a) . Monomers form 1D arrays within the P 4 2 2 1 2 crystals of TbCatB assembled in vivo .…”