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NMR应用于大型蛋白质时，所得到的光谱缺乏信号强度和分辨率。这个分辨率问题减少了可以通过核磁共振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。诸如蛋白质氘化以及TROSY和甲基-TROSY核磁共振脉冲序列等技术已经开发出来，以提高大蛋白质的核磁共振光谱的分辨率。虽然这些发展证明是成功的，但它们并不能解决问题。这个研究项目的目的是进一步发展这些技术来研究更大的蛋白质。目前最好的技术是甲基trosy，它通过用13CH3基团标记蛋白质而起作用。13CH3基团内不同偶极弛豫机制和化学位移各向异性弛豫机制之间的弛豫干扰。这导致核磁共振光谱中的每一个四重奏峰具有不同的线宽和强度，称为TROSY效应。相位循环可以用来绘制光谱中最尖锐、最强烈的峰，从而提高光谱的分辨率。氟原子核周围有一个大的不对称电子分布，导致一个大的CSA。因此，我们认为13CF3原子核会导致更大的弛豫干扰，从而增强核磁共振信号强度和分辨率。詹姆斯的研究将集中于核磁共振弛豫率的建模，随后表达13CF3标记的蛋白质，看看这种标记是否可以提高核磁共振光谱分辨率。虽然之前尝试测量13CF3标记的蛋白质中的TROSY效应是不成功的，但这些都是在较短的肽链中。在数学模型中，已经预测到较大的蛋白质会存在TROSY效应，但这需要实验证明。如果在13CF3标记的蛋白质中观察到TROSY效应，并且效果比13CH3标记的蛋白质更强，那么通过NMR研究大型蛋白质的新方案将被开发出来，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，而不会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，其核磁共振光谱目前遭受低信号强度和分辨率。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.