The use of 13CF3 nuclear spin probes to reduce the size limit of NMR spectroscopy.

核磁共振波谱是唯一可以确定蛋白质在溶液状态下的结构和动力学的技术之一。然而，当核磁共振应用于大型蛋白质时，所产生的光谱存在信号强度和分辨率不足的问题。这个分辨率问题减少了核磁共振可以有效研究的蛋白质的数量，这意味着关于它们动力学的重要信息被遗漏了。为了提高大蛋白质的核磁共振谱的分辨率，已经开发了诸如蛋白质重氢以及TROSY和甲基-TROSY核磁共振脉冲序列等技术。虽然这些发展被证明是成功的，但它们并没有能够解决问题。这项研究项目的目的是进一步发展这些技术，以研究更大的蛋白质。目前最好的技术，甲基-TROSY，是通过标记13CH3基团的蛋白质来工作的。13CH3基团中不同偶极和化学位移各向异性(CSA)弛豫机制之间的弛豫干扰。这导致核磁共振谱中四重组峰的每个峰具有不同的线宽和强度，称为TROSY效应。然后，可以使用相位循环来仅绘制光谱中最尖锐、最强烈的峰，从而提高光谱的分辨率。氟核周围有很大的不对称电子分布，导致了较大的CSA。因此，人们认为13CF3核将产生更大的弛豫干扰，从而提高核磁共振信号的强度和分辨率。詹姆斯的研究将集中在核磁共振弛豫速率的模拟上，然后是表达13CF3标记的蛋白质，看看这种标记是否可以提高核磁共振谱分辨率。虽然以前在13CF3标记的蛋白质中测量TROSY效应的尝试没有成功，但这些都是在更短的肽链中进行的。在数学模型中，已经预测对于较大的蛋白质将存在TROSY效应，但这需要实验证明。如果对13CF3标记的蛋白质观察到TROSY效应，并且这种效应比13CH3标记的蛋白质更强，那么通过核磁共振研究大蛋白质的新方法将被开发出来，显著提高信号强度和分辨率。使用13CF3探针的另一个好处是，在生物学中很少发现19F核，这意味着可以在体内研究感兴趣的蛋白质，而不需要受到背景噪声的影响。然后，该方案可以应用于一系列大型生物相关蛋白质，这些蛋白质的核磁共振谱目前存在信号强度和分辨率较差的问题。因此，可以确定这些蛋白质的重要结构和动力学。

NMR spectroscopy is one of the only techniques that can determine the structure and dynamics of a protein in the solution state. However, when NMR is applied to large proteins, the resulting spectra suffer from a lack of signal intensity and resolution. This resolution problem has reduced the number of proteins that can be studied effectively by NMR, meaning that important information on their dynamics is being missed. Techniques such as protein deuteration as well as TROSY and methyl-TROSY NMR pulse sequences have been developed to increase the resolution in the NMR spectra of large proteins. While these developments have proved successful, they have not been able to solve the problem. The purpose of this research project is to develop these techniques further to study even larger proteins. The current best technique, methyl-TROSY, works by labelling proteins with 13CH3 groups. Relaxation interference between different dipolar and chemical shift anisotropy (CSA) relaxation mechanisms within the 13CH3 group. This leads to each peak of the quartet in the NMR spectrum having a different linewidth and intensity, known as the TROSY effect. Phase cycling can then be used to plot only the sharpest, most intense peak in the spectrum, enhancing the resolution of the spectrum. Fluorine nuclei have a large asymmetric electron distribution around them, leading to a large CSA. Therefore, it is thought that 13CF3 nuclei will lead to greater relaxation interference enabling enhanced NMR signal intensity and resolution. James' research will focus on the modelling of NMR relaxation rates followed by expression 13CF3 labelled proteins to see if this labelling can improve the NMR spectrum resolution. Whilst previous attempts to measure the TROSY effect in 13CF3 labelled proteins have been unsuccessful, these have been in shorter peptide chains. In mathematical models, it has been predicted that the TROSY effect will exist for larger proteins, but this needs to be proved experimentally. If the TROSY effect is observed for 13CF3 labelled proteins and the effect is stronger than for 13CH3 labelled proteins, a new protocol to study large proteins via NMR will have been developed, significantly increasing signal intensity and resolution. Another benefit of using a 13CF3 probe is that 19F nuclei are rarely found in biology, meaning that the protein of interest could be studied in-vivo without the spectrum suffering from background noise. This protocol could then be applied to a range of large biologically relevant proteins whose NMR spectra currently suffer from poor signal intensity and resolution. Thereby, important structures and dynamics of such proteins can be determined.