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使用贫13C葡萄糖作为微生物生长碳源进行基于核磁共振的配体结合研究的低13C背景:13C标记的葡萄糖和13C-福司可林与大肠杆菌中半乳糖-H+同向转运蛋白GalP的结合

Low 13C-background for NMR-based studies of ligand binding using 13C-depleted glucose as carbon source for microbial growth: 13C-labeled glucose and 13C-forskolin binding to the galactose-H+ symport protein GalP in Escherichia coli.

作者信息

Patching Simon G, Herbert Richard B, O'Reilly John, Brough Adrian R, Henderson Peter J F

机构信息

Astbury Center for Structural Molecular Biology and Institute of Materials Research, University of Leeds, Leeds LS2 9JT, UK.

出版信息

J Am Chem Soc. 2004 Jan 14;126(1):86-7. doi: 10.1021/ja038275c.

Abstract

Obtrusive 13C-backgrounds can be a problem in 13C NMR-based studies of ligand binding to bacterial membrane transport proteins in their natural state in inner membranes. This is largely solved for the bacterial galactose-H+ symport protein GalP by growing the producing organism Escherichia coli on 13C-depleted glucose (13C </= 0.07%) as the main carbon source. 13C solid-state NMR-based binding studies for the inhibitor forskolin 1 and the transported substrate glucose 2, both singly labeled with 13C, are reported and discussed. For 1, tight binding is observed, while for 2, significant exchange takes place during the time scale of the NMR experiment.

摘要

在基于¹³C核磁共振(¹³C NMR)的研究中,当在内膜中研究配体与细菌膜转运蛋白的天然状态结合时,¹³C背景信号干扰可能会成为一个问题。对于细菌半乳糖-H⁺同向转运蛋白GalP,通过将产蛋白的大肠杆菌培养在以¹³C贫化葡萄糖(¹³C≤0.07%)作为主要碳源的培养基上,这个问题在很大程度上得到了解决。本文报道并讨论了基于¹³C固态核磁共振的结合研究,该研究针对抑制剂福斯可林1和转运底物葡萄糖2,二者均用¹³C单标记。对于1,观察到紧密结合,而对于2,在核磁共振实验的时间尺度内发生了显著的交换。

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