ABSTRACT
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.
Subject(s)
Manganese , Muramidase , Muramidase/chemistry , Manganese/chemistry , Crystallography, X-Ray , Porosity , Coordination Complexes/chemistry , Models, Molecular , Animals , Carbon Monoxide/chemistry , Time Factors , ChickensABSTRACT
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.
