Measuring nanometre distance changes in biomolecules under pressure

生物分子，如蛋白质，通过其结构排列的变化来发挥作用。观察和了解这些变化可以详细阐明生化机制，这反过来又可以导致纳米技术和药物发现等领域的进步。使用脉冲EPR技术，双电子-电子共振，鹿(PELDOR)，可以测量特定放置的自旋标签分子对之间的纳米级距离。移动这些标记在蛋白质中的位置可以使其结构被绘制出来。除了距离，还可以确定这些标记的相对取向，这可以提供关于蛋白质结构的进一步信息(G.Jeschke，Annu)。菲斯牧师。化学。(2012)63,419-446)。虽然蛋白质在生理条件下通常以有序的自然状态存在，但它们的功能可能会导致它们转移到激发状态。由于这些态具有较高的能量、较低的平衡布居和构象柔性，因此不能用传统的光谱技术来测量激发态。有趣的是，这可以通过将样品置于高压下来克服，这可能导致蛋白质的结构状态发生变化，并可以填充激发状态(K.Akasaka，生物化学)。(2003年)42,10875-10885)。将其与定点自旋标记和鹿光谱学相结合，可以允许访问这些状态，如果样本可以快速冷冻的话(M.T.Lerch等，Proc.娜塔莉。阿卡德。SCI。(2014)111，E1201-E1210)。在本工作中，我们将把这种方法应用于信使蛋白钙调蛋白，它随着钙浓度的变化以及结合蛋白的存在而发生结构变化。我们计划使用我们自制的HiPER(P.A.S.Cruickshank等人，Rev.Sci)光谱仪在Q波段和W波段进行测量。Instrum。(2009年)80,103102)。商定的培训要求是学习最佳研究实践、出版物和论文的科学写作，以及学习各种研究技术。除此之外，还需要进一步的学术发展，形式包括QM-CDT培训单元、教授的SUPA课程、出席每周座谈会和可转让的技能课程。此外，还应参与公众参与和教学。

Biomolecules, such as proteins, function by means of changes in their structural arrangement. Observing and understanding these changes can allow for detailed elucidation of biochemical mechanisms, which in turn can lead to advances in fields such as nanotechnology and drug discovery. Distances on a nanometre scale can be measured between pairs of specifically placed spin-label molecules using the pulsed EPR technique, double electron-electron resonance, DEER (PELDOR). Moving the locations of these labels within a protein can enable its structure to be mapped. As well as distances, the relative orientation of these labels can also be determined which can provide further information on the structure of the protein (G. Jeschke, Annu. Rev. Phys. Chem. (2012) 63, 419-446).While proteins typically exist in well-ordered native states under physiological conditions, their functions may cause them to move to excited states. Due to these states having higher energies, lower equilibrium populations and conformational flexibility, the excited states cannot be measured by traditional spectroscopic techniques. Interestingly, this can be overcome by subjecting the sample to high pressure, which can result in changes to the structural state of the protein and can populate the excited state (K. Akasaka, Biochemistry. (2003) 42, 10875-10885). Combining this with site-directed spin labelling and DEER spectroscopy can allow for these states to be accessed, if the sample can be rapidly frozen (M.T. Lerch et al, Proc. Natl. Acad. Sci. (2014) 111, E1201-E1210).In this work, we will apply this methodology to the messenger protein calmodulin which undergoes structural changes both with the calcium concentration as well as with the presence of binding proteins. We plan to measure at Q-band frequency and also at W-band using our home-built spectrometer, HiPER (P.A.S. Cruickshank et al, Rev. Sci. Instrum. (2009) 80, 103102). Agreed training requirements are to learn best research practice, scientific writing for publications and thesis, and to learn various research techniques. On top of these, further academic development is required in the form of QM-CDT training blocks, taught SUPA courses, attendance at the weekly colloquium, and transferrable skill courses. Further, involvement in public engagement and teaching is expected.