Shining Light on Metalloprotein Mechanism: Single Protein Crystal Catalytic Studies Driven by 'Caged' Electron Sources
Lead Research Organisation:
University of Leicester
Department Name: Chemistry
Abstract
Early reviews of time-resolved crystallography identified the need for generalised ways of triggering reactivity. Roughly 30-50% of proteins are redox proteins, one third of all proteins contain a redox-active metal, and approximately 22% of submissions to the PDB contain a transition metal, so new methods that enable time-resolved study of redox reactions using sub-turnover techniques will have significant academic impact. Pulse radiolysis or X-ray photoreduction are not generally for protein studies, causing primary and secondary radiation damage and leading to structural ambiguity in reduced states. The methods proposed here use lower energy triggers; we anticipate future use of longer wavelength chromophores, further minimising risk of photodamage.
The ambitious technical developments in this proposal have the potential to revolutionise biophysical capabilities, enabling studies of redox protein mechanism in exquisite chemical and structural detail. Combining single crystal spectroscopy, electrochemical control, and synchronous reaction initiation using a 'photo-caged' electron source we will build a platform technology with potentially transformative impact on biophysics and structural biology, and provide unprecedented possibilities to exploit time-resolved crystallographic and spectroscopic methods at national and international facilities. Thus far these methods have been largely inaccessible to 'real time' studies of redox proteins, as generalised methods to synchronise redox reactivity in the crystalline state do not exist. The methodology developed here overcomes the challenges of rapid triggering of electrochemical reactions in crystallo, whilst simultaneously allowing in situ infrared spectroscopic monitoring of transient redox species to characterise electrocatalytic reactions on sub-turnover timescales. This cutting-edge enabling technology will allow studies of previously inaccessible catalytic intermediates, driving scientific progress in biophysics, chemical and structural biology, and establishing the UK at the forefront of these unique and exciting scientific developments.
The ambitious technical developments in this proposal have the potential to revolutionise biophysical capabilities, enabling studies of redox protein mechanism in exquisite chemical and structural detail. Combining single crystal spectroscopy, electrochemical control, and synchronous reaction initiation using a 'photo-caged' electron source we will build a platform technology with potentially transformative impact on biophysics and structural biology, and provide unprecedented possibilities to exploit time-resolved crystallographic and spectroscopic methods at national and international facilities. Thus far these methods have been largely inaccessible to 'real time' studies of redox proteins, as generalised methods to synchronise redox reactivity in the crystalline state do not exist. The methodology developed here overcomes the challenges of rapid triggering of electrochemical reactions in crystallo, whilst simultaneously allowing in situ infrared spectroscopic monitoring of transient redox species to characterise electrocatalytic reactions on sub-turnover timescales. This cutting-edge enabling technology will allow studies of previously inaccessible catalytic intermediates, driving scientific progress in biophysics, chemical and structural biology, and establishing the UK at the forefront of these unique and exciting scientific developments.
People |
ORCID iD |
Philip Ash (Principal Investigator) |
Description | Through this award, I have developed a novel research method for studying enzymes that carry out chemistry involving the transfer of electrons. These new methods have resulted in collaboration with the Central Laser Facility. A particularly noteworthy finding involves new insight into how protons (H+) and electrons 'move' in biology, providing evidence that these events are not always concerted processes. These finding open up new avenues of research for the diverse array of proteins and enzymes involved in electron transfer reactions. |
Exploitation Route | The methodology developed will be of broad interest to researchers working in energy materials, metalloproteins, and chemical catalysis. The results obtained have the potential to spark new insight and research directions into biological energy transfer mechanisms and structural biology in particular. |
Sectors | Chemicals Energy Environment Other |
Description | Significant academic interest has been generated through this project, leading to potential new multidisciplinary research areas at the interface between physical and life sciences. The work represents a new breakthrough in understanding of energy transfer during biological redox catalysis, that has potential applications beyond the biological sciences in chemical catalysis and energy materials, for example. |
First Year Of Impact | 2023 |
Impact Types | Policy & public services |
Description | STFC Life Sciences and Soft Materials Advisory Group |
Geographic Reach | National |
Policy Influence Type | Participation in a guidance/advisory committee |
URL | https://www.ukri.org/who-we-are/stfc/how-we-are-governed/advisory-boards/life-sciences-and-soft-mate... |
Description | NextGen Structural Biology under Electrochemical Control: Filling in Missing Intermediates in Metalloenzyme Catalytic Cycles |
Amount | £1,131,075 (GBP) |
Funding ID | BB/X002292/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2023 |
End | 12/2025 |
Title | Nanosecond transient IR spectrometer |
Description | We have developed an infrared microscope using a tuneable QCL source for the purpose of measuring nanosecond-millisecond kinetics of metalloenzymes in solution. The instrument interfaces with an electrochemical cell using minigrid electrodes allowing electrocatalytic reactions of metalloenzymes to be studied. Reactions are initiated by a ns visible light trigger. |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2022 |
Provided To Others? | No |
Impact | Creation of the method has led to follow-on funding from BBSRC as noted elsewhere. |
Description | SpitFire Laser Loan |
Organisation | Rutherford Appleton Laboratory |
Department | Central Laser Facility |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Experimental design expertise, with collaboration formed during during competitively-awarded experimental time at the ULTRA facility (several publications in progress). |
Collaborator Contribution | Long-term loan of an ultrafast laser system and optical tables in order to set up a complementary research facility at the University of Leicester. |
Impact | A total of six weeks of collaborative experimental time have been awarded as a result of this collaboration. Multidisciplinary research spanning EPSRC, STFC, and BBSRC. |
Start Year | 2023 |
Description | Diamond Light Source PEER Review Panel |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Joined Peer Review Panel 5 (from March 2024), assessing user applications to Diamond Light Source and taking part in allocation of beamtime. |
Year(s) Of Engagement Activity | 2024 |
URL | https://www.diamond.ac.uk/Users/Apply-for-Beamtime/Peer-Review.html |
Description | Membership of Royal Society of Chemistry Committee |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Membership of committee for Inorganic Biochemistry Discussion Group, responsible for awarding prizes, conference organization, and promotion of bioinorganic chemistry within the UK and globally. Part of Royal Society of Chemistry. |
Year(s) Of Engagement Activity | 2023,2024 |
Description | School visits (Leicestershire) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Online and in-person delivery of research-themed talks to local schools, 6 talks to approx. 30 students each, lead to a discussion about my career in science and spectroscopy-themed activity. |
Year(s) Of Engagement Activity | 2021,2022,2023,2024 |