Heaven Graham, Hollas Michael A, Tabernero Lydia, Fielding Alistair J
Department of Chemistry, The University of Manchester, Manchester, M13 9PL UK.
School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, M13 9PL UK.
Appl Magn Reson. 2021;52(8):959-970. doi: 10.1007/s00723-021-01350-1. Epub 2021 Jun 10.
Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron-electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to 'knock out' superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies.
The online version contains supplementary material available at 10.1007/s00723-021-01350-1.
蛋白质及其生物复合物的结构研究现在经常通过使用双电子-电子共振（DEER）光谱法，通过定点自旋标记（SDSL）在自旋标记的半胱氨酸之间产生的距离限制来补充。甲硫基磺酸盐自旋标记（MTSSL）在蛋白质的SDSL中已变得无处不在，然而，由于其大量的旋转异构体和可还原性而存在局限性。在本文中，我们介绍了使用溴丙烯醛自旋标记（BASL）作为半胱氨酸自旋标记，证明了其相对于MTSSL的优势，因为它对表面半胱氨酸的选择性增加，无需“敲除”多余的半胱氨酸残基。应用于多结构域蛋白组氨酸结构域蛋白酪氨酸磷酸酶（HD-PTP），我们表明BASL可以通过选择性标记轻松过量添加，而MTSSL会导致蛋白质沉淀。此外，使用DEER，我们能够测量HD-PTP中三个半胱氨酸结构域内的单个半胱氨酸对距离。该标记还有一个进一步的优势，即在三键连接中包含一个硫化物，使其成为蛋白质结合和细胞内研究的候选物。
在线版本包含可在10.1007/s00723-021-01350-1获取的补充材料。
