Cryogen-free arbitrary waveform EPR for structural Biology and Biophysics

With the ever-growing popularity of site-directed mutagenesis and site-specific spin labelling EPR has become an established biophysical tool for tracing structural or conformational transitions and quaternary complex formation. The increasing complexity of the systems studied multiplies the demand on the sensitivity and accuracy of the EPR methods used. As a response, there is rapid development of novel labelling strategies and of more intricate experiments that demand full control of the frequency, phase and amplitude profiles of the microwave irradiation for EPR excitation. The ability to engineer arbitrary pulses is critical to new enabling experiments, designed to improve sensitivity and resolution by precisely controlling the excitation of spins. Additionally, most advanced EPR experiments require cryogenic cooling to achieve meaningful results on biological samples. Today, nearly all new EPR installations in the UK are equipped with cryogen-free (cf) cooling systems to remove the costly and inconvenient use of liquid helium. These cf systems provide better control of temperature, permitting longer experiments and reduce safety hazards related to liquefied gases. This proposal seeks to fund arbitrary waveform generator and cryogen-free cryostat upgrades for a commercial Bruker E580 X-band pulse EPR spectrometer at the Biomedical Sciences Research Complex, St Andrews. The projects that would greatly benefit from the new technology are involving nanometre distance measurements between radical spin-labels and/or paramagnetic metal ions and the characterisation of intrinsic or engineered paramagnetic sites and of their surroundings by pulse EPR and hyperfine spectroscopy. Current systems are ranging from membrane proteins to nucleic acids, covering research topics as diverse as mechanosensation and genome maintenance.