Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.

生物物理技术是探索生物过程的本质和所涉及的生物分子的有力途径。电子顺磁共振（EPR）光谱仪能够提取有关这些生物分子中包含未配对电子的原子周围局部原子环境的精细详细信息-我们将这些电子称为自由基或顺磁中心。生物分子如蛋白质可能含有天然的顺磁中心，如铜或铁，这通常是蛋白质工作的基础，或者实验人员可能会将顺磁金属或自旋标签附加到蛋白质的特定部分作为分子“间谍”。这些分子间谍可以用来测量局部动力学或测量在顺磁中心对之间约10纳米的区域内的远程纳米尺度距离。这是通过使用脉冲EPR测量这些对之间的磁相互作用来实现的，这类似于测量两个条形磁铁之间的力（因此高度依赖于它们的分离距离）。距离和距离分布都可以找到。这些实验的例子是双电子电子共振（DEER）和弛豫诱导的偶极调制增强（RIDME），这些技术统称为脉冲偶极光谱（PDS）。这种能力已被证明对研究各种生物大分子的结构和相互作用非常有用，现已成为生物分子研究的标准工具。在过去的10年里，这也是一个技术进步巨大的领域，灵敏度提高了一个多数量级，这是革命性的。更高频率和更高功率的光谱仪已经取得了进步，我们以前的“最先进的”商业主力“q波段”光谱仪几乎可以连续工作。然而，快速数字电子学的最新进展意味着灵敏度和样品吞吐量的进一步显著增加已经成为可能。这在很大程度上来自于在复杂序列中塑造微波脉冲的相位、频率和幅度的能力。对于许多实验，这减少了10倍的测量时间，大大提高了多用户，多项目设施的使用和能力，专注于生物应用。我们和几乎所有领先的EPR专家都将这项技术视为生物科学EPR技术的未来。我们有大量的潜在用户和各种各样的生物系统，这些系统将首次成为可询问的。我们的研究人员、合作者和合作伙伴来自广泛的国家和国际机构。我们在该领域有着广泛的记录，相信这次升级将大大提高英国在生物EPR方面的能力和声誉。该提案将确保我们位于圣安德鲁斯/邓迪的中心的可持续性。我们的技术和应用经理的职位将在未来三年得到保证，我们15年的光谱仪将获得新生。这些改进将使我们保持国际竞争力，并继续发展和应用EPR技术解决生物科学领域的相关问题。

Biophysical techniques are a powerful way to explore the nature of biological processes and the biological molecules involved. The electron paramagnetic resonance (EPR) spectrometer is capable of extracting exquisitely detailed information about the local atomic environment surrounding atoms within these biological molecules that contain unpaired electrons - we refer to these as radicals or paramagnetic centres. Biological molecules such as proteins may contain natural paramagnetic centres, such as copper or iron, which are often fundamental to how the protein works, or an experimenter may incorporate paramagnetic metals or attach spin labels to specific parts of proteins as a molecular "spy".These molecular spies can be used to measure local dynamics or measure long-range nanometre scale distances in the region to about 10 nm between pairs of paramagnetic centres. This is achieved by measuring the magnetic interaction between these pairs using pulsed EPR, which is analogous to measuring the force between two bar magnets (and is therefore highly dependent on their separation distance). Both distances and distance distributions can be found. Examples of these experiments are double electron electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME) and collectively the techniques are referred to as pulsed dipolar spectroscopy (PDS).This ability has proved very useful to the study of the structure and interactions of a wide variety of biomacromolecules and has now become a standard tool in biomolecular research. It is also a field which has seen tremendous technical advances over the last 10 years with sensitivity increasing by more than an order of magnitude, which has been transformative. Advances have come from higher frequency and higher power spectrometers, and our previous "state-of-the-art" commercial work-horse "Q-band" spectrometer operates almost continuously. However recent advances in fast digital electronics mean that further significant increases in both sensitivity and sample throughput have become possible. A large part of this comes from the ability to shape the phase, frequency and amplitude of microwave pulses in complex sequences. For many experiments this reduces measurement time 10-fold, dramatically increasing usage and capability for a multi-user, multi-project facility focussed on biological applications. We, and nearly all leading EPR experts, see this technology as the future of the EPR technique for the biosciences. We have a large base of potential users and a wide variety of biological systems that would become interrogatable for the first time. Our investigators, collaborators and partners come from a wide range of national and international institutions. We have an extensive track record in the field and believe this upgrade will substantially increase the UK's capability and reputation in biological EPR. This proposal will ensure sustainability for our centre based at St Andrews/Dundee. The position of our technical and applications manager will be secured for a further three years, and our fifteen-year-old spectrometer will be given a new lease of life. These improvements will allow us to remain internationally competitive and to continue developing and applying the EPR technique to relevant problems across the biosciences.