2023 Fiscal Year Research-status Report
Design of protein crystal templates with multiple binding sites for tracking metal complex reactions.
| Project/Area Number |
23K04928
|
|---|
| Research Institution | Tokyo Institute of Technology |
|---|
Principal Investigator |
Maity Basudev 東京工業大学, 生命理工学院, 特任助教 (60815421)
|
|---|
| Project Period (FY) |
2023-04-01 – 2026-03-31
|
| Keywords | RNaseA crystal / Mn-carbonyl comples / CO release reaction / X-ray crystal structure |
|---|
| Outline of Annual Research Achievements |
We used RNaseA as a template to study a reaction in multiple protein environments. We immobilized a Mn-carbonyl complex in RNaseA crystal by soaking. The X-ray crystal structure revealed four different metal binding sites which are located at N-terminal, Asp53, His105 and His119. In all the cases, the density of three CO ligands were observed at the Mn center which showed the characteristic CO stretching frequency at 1931 cm-1. Then, we performed the CO release reaction by exposing the single crystal in UV light. The reaction at Asp53 and His105 was very fast and after 10 min of irradiation, all the CO were released whereas other sites required longer time. This showed the direct effect of protein environment in a reaction.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
The reaction is progressing smoothly as we planed. This is because our proposed work was based on some preliminary result (X-ray structure) and then we optimize the metal immobilization and determination of high resolution X-ray structure. Since we obtained the high-resolution X-ray structure, it became easy to understand each reaction center and the local protein environment. Currently, we are focusing on the next step to study the details of CO reaction within the protein crystal.
|
| Strategy for Future Research Activity |
As a next plan, we are aiming to study the detail CO release reaction within the protein crystal. We already obtained the preliminary data after the light irradiation. Therefore, we first focus on optimizing the light exposure time to capture the intermediates during the CO release reaction. In addition, we also plan to add coordinating ligand such as imidazole in the buffer solution which are expected to bind to Mn after the release of CO. This will give additional information about the trapped intermediate. We also plan to perform QM calculation to validate the experimental results.
|
| Causes of Carryover |
This is because the proposed research progressed well and we need to measure the X-ray structure at SPring8 quickly in the next beamltime.
|
