A nanosecond laser spectroscopy platform for studying light-activated biomolecules

There are many processes in biology that are triggered by light (e.g. photosynthesis, photoreceptor proteins, light-activated enzymes or engineered systems). Many of these changes induced by exposure to light occur across multiple timescales (typically from fs - s), and involve complex reaction cascades of chemical and structural change in both chromophores and the biological macromolecule (typically protein). This requires comparative analysis of photophysical, photochemical and structural change across a wide range of timescale. Through previous BBSRC funding we have customised and built a range of field-leading time-resolved spectroscopy instruments on ultrafast (sub-nanosecond) timescales complete with visible and infra-red absorbance and fluorescence detection capabilities to interrogate complex light-driven mechanisms. Although light-sensitive biological processes require excited state dynamics on these ultrafast timescales the photochemical and structural changes that drive biological function generally occur on slower timescales (nanosecond-second). Monitoring these processes is crucial to provide important insight into mechanisms and informs rational re-design.

We now propose a multi-purpose time-resolved spectroscopy instrument to support a wide range of programmes in the UK bioscience community. The instrumentation will have a wide range of capabilities that uniquely includes detection in both the UV-visible and infra-red regions. The proposed technology platform comprises a nanosecond laser system that will be used to trigger 2 separate time-resolved spectrometers, one that can measure transient absorbance changes in the UV-visible region and the other in the infra-red region, both on the nanosecond-millsecond timescale. This novel experimental set up, comprised of truly 'plug and play' components, will allow us to correlate changes in the UV-visible with those in the mid-infra-red region where biological systems have many vibrational modes. This instrumental configuration will be unique to the UK bioscience community and will provide significant infrastructure and technology resource for many research groups at the Manchester institute of Biotechnology, the wider University of Manchester and external collaborators, where a number of research projects have been identified. The integrated facility will be a community-based advanced instrument, supported by expert experimental scientists and internationally recognised academic leadership.

In light-activated systems, a laser pulse can be used to initiate the reaction, and another synchronised pulse used to monitor the subsequent changes in electronic and vibrational energy levels. In nanosecond time-resolved spectroscopy techniques a probe in either the UV/visible or IR region is electronically delayed relative to a short laser pulse (typically 3-5 ns) to provide high signal:noise and fast time resolution detection. The laser system central to the proposed facility (a Nd:YAG laser system and associated OPO to produce wavelengths ranging from 405 to 2600 nm) will be used to pump 2 time-resolved spectrometers, a laser flash photolysis instrument which probes the UV/visible region, and an EOS IR infra-red broadband pump-probe transient absorption spectrometer, which probes the infra-red region where biological systems have many vibrational modes. The UV-visible laser flash photolysis instrument uses a pulsed Xenon arc lamp as the probe and uniquely, it also comes equipped with a CCD detector to allow measurements of the whole absorbance spectrum at defined time points. Moreover, the provision of a stopped-flow accessory and additional temperature controlled sample holders ensures maximum flexibility in the range of possible experiments. The EOS IR instrument builds on previous BBSRC funding in this area as it shares many components with the Helios IR instrument that was purchased with ALERT 14 funding to cover the entire time window from picoseconds to milliseconds, making the integration extremely cost effective. This facility will allow us to probe photo-induced changes in UV/visible and IR absorption from ns - s using identical excitation conditions in a single technology platform, which will provide a detailed picture of the photodynamics and ensures an efficient use of biological samples. In addition, the ability to probe liquid, solid, or even crystalline samples extends the scope of the facility to cover almost every imaginable system.

We will establish a multi-purpose time-resolved spectroscopy instrument requested in this funding application will provide a unique platform for studying light-activated biomolecules and will support a wide range of modern UK bioscience research programmes. The proposed time-resolved spectroscopy facility will be located in the Manchester Institute of Biotechnology close to biological research laboratories and managed by expert technical staff so that it would benefit a wide range of users (expert and non-expert) from across the disciplines.

The research that will be enabled by the acquisition of this new facility will have benefit to: (i) new and existing academic researchers who wish to study light-activated biomolecules as part of their wider research base, in particular across the remits of the BBSRC science committees; (ii) governmental and private sector scientists especially those engaged in using light-driven approaches to develop new capabilities in the biotechnology sector; (iii) international organisations including collaborators from academia, the public and private sectors.

In order to allow the instrumentation to be fully exploited we would ensure that the equipment was fully accessible to all UK academics, who would also benefit from the high level of expertise and training that will be available at Manchester. Access to both internal and external users would be managed by the supporting Senior Experimental Officers (Dr Heyes and Dr Hardman) who will also implement cost recovery mechanisms for open access.

The impact of the multi-user facility would be maximised by: (i) organising a project/facility specific workshops around the wider biophysics capabilities available in the MIB; (ii) generation of a web-portal for work conducted and opportunities for new investigators to the discipline; (iii) including details of the instrumentation on the N8 Shared Research Equipment Inventory to network with industry and via equipment.data.ac.uk; (iii) continuing our industrial collaborations; (iv) presentations to the scientific community via conferences and publications; (v) public engagement.

We will measure the success of our impact activities by the number of: (i) registered users of our community website/podcasts and associated outreach materials; (ii) applications to use the facility; (iii) scientific publications and citations thereof relating to the facility; (iv) number of new grant funded projects generated by the facility; (v) training of the next generation bioscientists using time-resolved spectroscopy in their wider research programmes; (vi) participants that attend our workshops and future collaborations with new external partners; (vii) new IP established through the facility.