Crack Jason C, Stewart Melissa Y Y, Le Brun Nick E
Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR47 TJ, UK.
Biol Methods Protoc. 2019 Jan 22;4(1):bpy015. doi: 10.1093/biomethods/bpy015. eCollection 2019.
The ability to specifically label the sulphide ions of protein-bound iron-sulphur (FeS) clusters with S isotope greatly facilitates structure-function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the -acetyl-l-serine sulfhydrylase CysK to generate S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply S for FeS cluster assembly in an otherwise standard cluster reconstitution protocol.
用硫同位素特异性标记蛋白质结合的铁硫（FeS）簇中的硫离子的能力极大地促进了结构-功能研究。特别是，当使用探测簇相关振动的光谱技术或非变性质谱时，它能提供有价值的信息，在非变性质谱中，每个硫的平均增加约+2 Da能够明确鉴定FeS簇及其相关的转化/降解产物。在这里，我们使用乙酰-L-丝氨酸巯基酶CysK的热稳定同源物来生成硫取代的L-半胱氨酸，随后将其用作L-半胱氨酸脱硫酶NifS的底物，以便在标准的簇重构方案中为FeS簇组装逐步提供硫。
