Hussain Rohanah, Harding Stephen E, Hughes Charlotte S, Ma Pikyee, Patching Simon G, Edara Shalini, Siligardi Giuliano, Henderson Peter J F, Phillips-Jones Mary K
Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, U.K.
National Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, Leicestershire, LE12 5RD, U.K.
Biochem Soc Trans. 2016 Jun 15;44(3):810-23. doi: 10.1042/BST20160023.
This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins-synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs.
本文综述了目前在大肠杆菌中进行可靠的异源过表达以及纯化毫克量属于双组分信号转导家族的整合膜蛋白细菌膜传感器激酶（MSK）的方法。这些方法中有许多是在利兹与史蒂夫·鲍德温教授共同开发的，谨以此文纪念他。本文还综述了两种我们已成功应用于纯化的MSK和其他膜蛋白研究的生物物理方法——同步辐射圆二色光谱（SRCD）和分析超速离心（AUC），这两种方法都是无需固定化和无基质的方法，不需要标记策略。其他技术如等温滴定量热法（ITC）也具有这些特点，但通常需要高浓度的材料。与许多其他生物物理技术一样，这两种生物物理方法都能提供有关膜蛋白构象、寡聚化状态和配体结合的信息，但它们还具有额外的优势，即能够直接评估配体结合相互作用是否伴随着构象变化。因此，这两种方法都提供了一种强大的手段，用于识别和表征抑制剂结合以及任何相关的蛋白质构象变化，从而为未来针对细菌MSK的药物干预策略提供有价值的信息。
