Award details

Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs

Reference	BB/W003066/1
Principal Investigator / Supervisor	Dr Robin Owen
Co-Investigators / Co-Supervisors	Dr Danny Axford
Institution	Diamond Light Source
Department	Science Division
Funding type	Research
Value (£)	357,199
Status	Current
Type	Research Grant
Start date	14/03/2022
End date	13/03/2025
Duration	36 months

Abstract

X-ray crystallography has been the leading method to understand structure and mechanisms of proteins for decades. However, the structures obtained generally represent averaged or static states and cannot fully represent dynamic proteins. Obtaining time resolved, high-resolution structures of proteins as they carry out their functions is a grand challenge for life science. Time resolved x-ray crystallography has largely been limited to reversible, naturally photoactivatable e.g. rhodopsins (a tiny fraction of all proteins), with more recent developments allowing access to non-reversible processes such as enzyme reactions. We will use laser activated photocages and silicon fixed targets to initiate reactions in microcrystals of proteins of fundamental and biotechnological importance. These include enzymes relevant to biofuel production (lytic polysaccharide monooxygenases) and gas sensor proteins (cytochromes c') and cytochrome P450Nor. Our approach using time-dependent x-ray serial crystallography will allow access to a very wide range of timescales, from milliseconds to minutes, allowing on-pathway intermediates to be characterised. E.g. catalytic intermediates arising from oxygen activation by 2 mononuclear copper centres will be identified along with capturing the elusive distal intermediate/structural reorganisation associated with binding in a range of haem gas sensor proteins. Time-dependent crystallographic data will be measured with reaction progress and identity of intermediates monitored by complimentary spectroscopies providing essential validation of structures and allowing maximum biological information to be obtained. Data will be measured using microfocus synchrotron and x-ray free electron laser facilities. The results obtained will provide important time-dependent and dynamic mechanistic information. Our research program will also establish a general method, addressing the grand challenge of acquiring time resolved structures of enzymes in action

Summary

Efforts to understand how enzyme catalysis and protein-ligand binding work (or how they fail to work properly) is a vital research field in the life sciences. The knowledge gained has significance for the development of efficient biosynthetic materials, for applications in the biochemical, biotechnology and industrial sectors and in developing new medicines. Obtaining the structures of enzymes is an essential part of this effort. X-ray crystallography is a technique that reveals the individual atoms that make up an enzyme molecule, showing how they are joined to each other to form larger 3D structures. These structures and how they change over time as an enzyme performs its work are key factors for our understanding of enzyme function. This 'time-resolved' aspect is analogous to moving from a single photograph to a movie, and is tremendously powerful but also very challenging to achieve. To uncover this detailed 3D arrangement of atoms within an enzyme, powerful X-ray sources, called synchrotron storage rings and X-ray free electron lasers (XFELs), are used to visualise crystallised versions of the enzyme, using micron sized X-ray beams. The structure of the enzyme can then be deduced from the way that the X-rays are 'diffracted' as they pass through the crystal. The crystals that are grown in the laboratory for these experiments, including many important pharmaceutical targets for therapies, are very often only available as tiny, micron-sized (one-thousandth of a millimetre) 'microcrystals'. We will use silicon 'chips' for efficient delivery to the X-ray beam, for optimum high-throughput and high 'hit rate' (a 'hit' is when the micron size X-ray beam is diffracted by a microcrystal). A chip is a device containing thousands of shaped wells each of which can trap a microcrystal. Each chip can hold up to 25,600 microcrystals and each well, with its trapped microcrystal, is exposed in turn to the X-rays beam at the selected experimental facility used (the Diamond synchrotron in the UK or SACLA in Japan). The different facilities used have very different properties so can be used to provide complementary information by for example probing different timescales. The X-ray diffraction patterns gathered from all the microcrystal hits are combined and analysed to create a 3D model (structure) of the enzyme. Using chips for sample delivery we can obtain a complete 3D structure of an enzyme in less than one hour, which is a very efficient use of these expensive X-ray facilities. We will first study enzymes in their 'resting' states before they undergo catalysis (do their work), giving us the initial structures of the enzymes. We will use oxygen and nitric oxide and photocages to initiate catalysis or ligand binding in microcrystals. Photocages are compounds that contain a trapped molecule of interest, which is held securely and only released by a brief flash of a laser beam. By collecting diffraction patterns from the microcrystals at varying time delays after the laser flash we can capture time dependent changes and build up an accurate molecular movie of the enzyme in action. Crucially, we are able to produce these structures at room temperature, close to the conditions under which enzymes function in nature. This allows their dynamic movements related to their function to be followed much more accurately compared to the cryogenic conditions (liquid nitrogen temperature, approximately -173 oC) at which crystal structures are typically produced. Our approach allows us to follow reactions over a wide timescale from microseconds to seconds or minutes, building up a complete picture of catalysis.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/W001950/1 Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs

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