Lunde Christopher S, Rouhani Shahab, Facciotti Marc T, Glaeser Robert M
QB3 Institute, University of California, Berkeley, CA 94720, USA.
J Struct Biol. 2006 Jun;154(3):223-31. doi: 10.1016/j.jsb.2006.02.002. Epub 2006 Mar 9.
Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.
在尝试对整合膜蛋白进行结晶时，蛋白质稳定性是一个需要考虑的关键因素。立方相或介观脂质双层结晶介质被认为能提供类似天然的环境，通过在结晶过程中帮助稳定天然蛋白质构象，从而促进膜蛋白结晶。虽然已经在由甘油单酯形成的脂质双层凝胶中获得了细菌视紫红质（bR）和其他嗜盐菌视紫红质的优质晶体，但此类介质在其他膜蛋白上的普遍应用仍难以成功。此外，我们注意到bR的一些突变体在由单油酸甘油酯形成的凝胶中高度不稳定。磷脂酰乙醇胺（PE）及其衍生物代表另一类可形成连接双层凝胶的脂质。当野生型bR和一个不稳定的bR突变体重构到这种磷脂凝胶中时，光谱显示与使用单油酸甘油酯形成的凝胶相比，该蛋白质更稳定且构象均一性有所改善。此外，我们证明可以从基于PE的结晶介质中生长出具有良好衍射能力的bR晶体。由于大多数蛋白质缺乏稳定性指示发色团，且其他基于结构的分析技术与脂质凝胶的兼容性较差，我们基于色氨酸残基的固有荧光开发了一种通用的光谱技术。这种荧光测定法使快速评估脂质凝胶作为膜蛋白结晶介质成为可能。
