Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

Displaying a Protein Cage on a Protein Crystal by In-Cell Crystal Engineering.

The development of solid biomaterials has rapidly progressed in recent years in applications in bionanotechnology. The immobilization of proteins, such as enzymes, within protein crystals is being used to develop solid catalysts and functionalized materials. However, an efficient method for encapsulating protein assemblies has not yet been established. This work presents a novel approach to displaying protein cages onto a crystalline protein scaffold using in-cell protein crystal engineering. The polyhedra crystal (PhC) scaffold, which displays a ferritin cage, was produced by coexpression of polyhedrin monomer (PhM) and H1-ferritin (H1-Fr) monomer in Escherichia coli. The H1-tag is derived from the H1-helix of PhM. Our technique represents a unique strategy for immobilizing protein assemblies onto in-cell protein crystals and is expected to contribute to various applications in bionanotechnology.

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

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.