Structure determination of the membrane bound protease LspA by NMR spectroscopy

脂蛋白信号肽II(LspA)是一种位于细菌膜上的细菌蛋白酶。LspA在前脂蛋白通过内膜输出后，将信号肽从前脂蛋白中分离出来。LspA属于一类特殊的天冬氨酸蛋白酶，在古生物或真核生物中不存在同源蛋白，这使其成为开发新抗生素的极佳靶点。该蛋白质含有164个氨基酸，预测有4个α-螺旋。我们希望通过液态核磁共振波谱确定LspA的结构，并研究环肽抑制剂球霉素如何与蛋白质相互作用。我们已经使用我们的无细胞表达系统建立了表达和标记方案，并在纳米盘上测量了LspA的2D和3D核磁共振谱，显示出非常好的化学位移分散性。所提供的数据表明，使用我们在过去建立的用于确定早老素和蛋白视紫红质的C-末端结构域的结构的方法，完全结构测定是可能的。

Lipoprotein signal peptidase II (LspA) is a bacterial protease located in the bacterial membrane. LspA cleaves the signal peptide from prolipoproteins after they are exported across the inner membrane. LspA belongs to an unusual class of aspartic acid proteases with no homologous proteins present in archaea or eukaryotes which makes it an excellent target for the development of new antibiotics. The protein contains 164 amino acids with four predicted alpha-helices. We want to determine the structure of LspA by liquid state NMR spectroscopy and investigate how the cyclic peptide inhibitor globomycin interacts with the protein. We have established expression and labelling protocols using our cell-free expression system and have measured 2D and 3D NMR spectra of LspA in nanodiscs that show very good chemical shift dispersion. The data presented suggest that a full structure determination is possible using the protocol that we have established in the past for the structure determination of the C-terminal domain of presenilin and of Proteorhodopsin.