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.

Elucidating Conformational Dynamics and Thermostability of Designed Aromatic Clusters by Using Protein Cages.

Multiple aromatic residues assemble to form higher ordered structures known as "aromatic clusters" in proteins and play essential roles in biological systems. However, the stabilization mechanism and dynamic behavior of aromatic clusters remain unclear. This study describes designed aromatic interactions confined within a protein cage to reveal how aromatic clusters affect protein stability. The crystal structures and calorimetric measurements indicate that the formation of inter-subunit phenylalanine clusters enhance the interhelix interactions and increase the melting temperature. Theoretical calculations suggest that this is caused by the transformation of the T-shaped geometry into π-π stacking at high temperatures, and the hydration entropic gain. Thus, the isolated nanoenvironment in a protein cage allows reconstruction and detailed analysis of multiple clustering residues for elucidating the mechanisms of various biomolecular interactions in nature which can be applied to design of bionanomaterials.

Engineering of an in-cell protein crystal for fastening a metastable conformation of a target miniprotein.

Protein crystals can be utilized as porous scaffolds to capture exogenous molecules. Immobilization of target proteins using protein crystals is expected to facilitate X-ray structure analysis of proteins that are difficult to be crystallized. One of the advantages of scaffold-assisted structure determination is the analysis of metastable structures that are not observed in solution. However, efforts to fix target proteins within the pores of scaffold protein crystals have been limited due to the lack of strategies to control protein-protein interactions formed in the crystals. In this study, we analyze the metastable structure of the miniprotein, CLN025, which forms a ß-hairpin structure in solution, using a polyhedra crystal (PhC), an in-cell protein crystal. CLN025 is successfully fixed within the PhC scaffold by replacing the original loop region. X-ray crystal structure analysis and molecular dynamics (MD) simulation reveal that CLN025 is fixed as a helical structure in a metastable state by non-covalent interactions in the scaffold crystal. These results indicate that modulation of intermolecular interactions can trap various protein conformations in the engineered PhC and provides a new strategy for scaffold-assisted structure determination.

Importance of the Subunit-Subunit Interface in Ferritin Disassembly: A Molecular Dynamics Study.

Ferritin is a spherical cage-like protein that is useful for loading large functional particles for various applications. To our knowledge, how pH affects the interfaces inside ferritin and the mechanism of ferritin disassembly is far from complete. For this article, we conducted a series of molecular dynamics simulations (MD) at different pH values to study how interfaces affect ferritins' stability. It is shown that dimers are stable even at extremely low pH (pH 2.0), indicating that the dimer is the essential subunit for disassembly, and the slight swelling of the dimer resulting from monomer rotation inside a dimer is what triggers disassembly. During ferritin disassembly, there are two types of interfaces involved, and the interface between dimers is crucial. We also found that the driving forces for maintaining dimer stability are different when a dimer is inside ferritin and in an acidic solution. At low pH, the protonation of residues can lead to the loss of the salt bridge and the hydrogen bond between dimers, resulting in the disassembly of ferritin in an acidic environment. The above simulations reveal the possible mechanism of ferritin disassembly in an acidic solution, which can help us to design innovative and functional ferritin cages for different applications.

Photoinduced in†Vivo Magnetic Resonance Imaging (MRI) with Rapid CO Release from an MnCO-Protein Needle Composite.

Construction of an artificial protein needle (PN), which includes the membrane puncturing needle domain of bacteriophage T4 conjugated to Mn carbonyl (MnCO) complexes, is reported. The responsiveness to visible light of the MnCO complex makes it useful as a photoinduced in vivo magnetic resonance imaging contrast reagent (MRI CR), because the PN carrier has the potential to deliver the MnCO complex into mouse tumors with retention of coordination structure within the in vivo environment. Moreover, the composite has higher relaxivity and longer circulation as an MRI CR than the corresponding MnCO complex. These results demonstrate construction of a responsive in vivo MRI CR by using an artificial metalloprotein.