The Research Complex at Harwell LIFEtime Instrument

The LIFEtime Instrument employs advanced laser based instruments to characterise changes in biologically important molecules such as proteins and DNA. These changes occur across a wide range of timescales and we will observe them using subtle changes in the way they absorb infrared laser light. The optical changes indicate changes in molecular structure and environment that underlie molecular reactivity. A wide range of BBSRC relevant research will be performed on the LIFEtime instrument in areas as diverse as protein folding, understanding photosynthesis, understanding the physical basis of catalysis and signalling in enzymes and light activated proteins and designing new biomolecular probes for cell imaging and medicine.

Most light driven processes in nature occur in cascades of gradually slowing steps; the faster ones affect the outcome of the slower ones. Thus, light harvesting that plants use to grow and produce food, for example, in photosynthesis starts by femtosecond processes, (1 femtosecond = a thousand million millionths of a second) and is the time scale of atoms moving in molecules. On these timescales primary energy and electron transfer (ET) reactions occur, yet are followed by hundreds of picoseconds (million millionths of a second) and longer times for molecular rearrangements that determine the yields of the reaction. The movement of electrons and protons within molecules is ultimately stabilised through separation of the negative and positive charges. This separation can be through diffusion or across a membrane taking place on microsecond to millisecond timescales. These longer time processes also include changes in molecular geometry, isomerisation, structural bond changes. Additional energy relaxation pathways include thermal processes and these also involve kinetic cascades such as in the case of protein folding, which starts with femtosecond structural fluctuations and finishes on millisecond timescale. We propose to install within the Research Complex at Harwell a world-unique instrument, LIFEtime, for interrogating kinetics and structural changes of biological systems spanning over 10 orders of magnitude timescale, within a single experiment, on a single sample and under identical experimental conditions. This will allow a comprehensive study of many dynamic processes in biological systems in their whole complexity.

The LIFEtime Instrument will utilise ultrafast lasers for Time-Resolved Multiple Probe Spectroscopy (TRMPS) to record sample changes across femtoseconds (fs) to milliseconds and beyond, as required, to follow the wide range timescales involved in natural processes.

TRMPS, an STFC-developed technique, relies on a dual laser system, one output used for triggering a sample change (pump) and a second output for measuring changes in the sample IR spectrum (probe). Pump pulses can use electronic/vibrational excitation or pH/temp jump to trigger the reaction of interest. The pump and probe have variable electronic and optical timing delay control, covering measurements in the fs to microsecond (us) timescale. By running the probe laser at high repetition rate (>=100 kHz), subsequent pulses following the single pump pulse can continue to probe the sample over longer times at <=10 us intervals, thus combining fast and slow measurements on one instrument. The instrument provides a highly reliable way to compare these radically different timescale events, which is otherwise impossible. This is also a highly efficient way of getting information from sensitive, precious samples, circumventing the need to repeat the experiment (which may involve irreversible sample damage) multiple times to observe the spectral changes over a range of timescales. The repetition rate of the pump is easily controlled to suit the sample of interest (high rate for samples which recover quickly or low for those with kinetics occurring over longer times).

Additionally, other advanced ultrafast spectroscopy techniques will be available, such as 2D-IR spectroscopy, providing detailed structural information through vibrational coupling. As high quality data will be available in seconds, measurements can be repeated indefinitely to measure structural changes over long timescales (e.g. seconds to hours).

The main beneficiaries of the LIFEtime instrument are the investigating research teams and their Project Partners, the residents of the Research Complex at Harwell (RCaH) through use of this shared instrument and collaboration with its users, the wider UK research community (see details below), RCUK and the UK.

The LIFEtime instrument will be unique, providing the UK with the best time-resolved infrared instrument for probing short to long timescale changes, using one instrument and one sample for all timescales, with "plug and play" ease of use. The ability of the proposed instrument to extend these studies to low concentration biological samples (thus reducing sample costs) will have a profound impact on the scientific community. Much of the research enabled by the instrument will follow changes in protein structure, is answering fundamental questions in natural processes which must be addressed in order to have impact in solving health problems through providing a greater understanding in protein/drug interaction.

The impact of this advanced instrument to the wider UK research community is evident from the accompanying letters of support. Provision for access to the machine by the wider community will be provided by setting aside 20% of available instrument time for their access. Fair access will be ensured by independent review through the Central Laser Facility's (CLF) Facility Access Panel, and this will encourage new collaborations and research teams studying in the bio-sciences under the BBSRC and Life Science interface remit. This operating model will ensure maximum impact through the delivery of the new technology as quickly as possible to a wide range of new users. A wider community still will be able to access the outputs of the research whether through publications, conference talks and/or press releases.

The location of the LIFEtime instrument in the highly multidisciplinary environment of the Harwell Oxford Campus and the RCaH will automatically encourage new collaborations and science programmes. The RCaH runs an active programme encouraging collaboration between its residents and visitors, and this programme will be extended to encompass the partners in this proposal and other users of the instrument. The RCaH also houses shared facilities used by users of all the main UK Facilities (Central Laser Facility, Diamond Light Source and ISIS) potentially opening up the instrument to a much larger user community.

Users of the instrument will have access to the STFC's resources to identify and exploit intellectual property (IP). The CLF has a prolific history of generating and securing IP. Pavel Matousek (Co-Investigator) has extensive experience in collaborating with industry (responsible for the Cobalt spin-out that stemmed from the Central Laser Facility's ULTRA research), and will co-ordinate economic impact activity in partnership with University Partners and STFC Innovations. To maximize benefit to all involved.

The instrument like all those housed within the Research Complex and the Central Laser Facility provide an excellent training opportunity for PhD students and users alike. Providing well trained researchers which will benefit the research community and potentially the UK economy.