Two-dimensional crystallization of a histidine-tagged protein on monolayers of fluidity-enhanced Ni2+-chelating lipids
Two-dimensional crystallization of a histidine-tagged protein on monolayers of fluidity-enhanced Ni2+-chelating lipids
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DOI：
10.1021/la026261z
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发表时间：
2002-11-26
期刊：
影响因子：
3.9
通讯作者：
Vénien-Bryan, C
中科院分区：
文献类型：
--
作者：
Courty, S;Lebeau, L;Vénien-Bryan, C
蛋白质在脂质单分子膜上的二维结晶是一种强有力的结构测定方法。这种方法已经扩展使用特定的和强烈的相互作用之间的组氨酸残基（过表达的蛋白质）和Ni 2+离子拴系在合成脂质的头基，理解，然后改善的过程中的吸附和结晶的蛋白质的脂质单层是生产大的和良好有序的晶体的任何可溶性或膜，组氨酸标记的蛋白质的先决条件。这些大型高质量阵列是高分辨率结构研究所必需的。我们已经研究了使用三种不同脂质的His-HupR的吸附和2D结晶的步骤：（i）2-（双羧甲基氨基）-6-[2-（1，3-二-O-油基-甘油氧基）-乙酰基-氨基]己酸镍-（II）（ii）Ni-NTA-BB，其具有两个支链（B）烷基链和（iii）Ni-NTA-BF，其为具有一个支链（B）和一个氟化（F）链的不对称脂质。这三种脂质在空气-水界面上铺展时，表现出各种流动性。吸附和结晶过程已监测原位带在真实的时间使用各种互补的技术，如椭圆偏振法，剪切刚度测量的单层，和布鲁斯特角显微镜，我们还开发了X射线：反射率分析，调查的电子密度分布的脂质-蛋白质单层的演变。还进行了蛋白质-脂质层的电子显微镜观察。我们已经发现，脂质单层的流动性对His-HupR的蛋白质吸附和结晶的速率有显着的影响。当Ni-NTA-BB用于形成单分子膜时，它加速了蛋白质吸附带的过程，蛋白质的结晶速度比Ni-NTA-DOGA快三倍。
Protein two-dimensional (2D) crystallization on lipid monolayers is a powerful method for structure determination. This method has been extended using the specific and strong interaction between histidine residues (of an overexpressed protein) and Ni2+ ions tethered at the headgroup of synthetic lipids, Understanding and then improving the process of adsorption and crystallization of proteins on a lipid monolayer are prerequisites for the production of large and well-ordered crystals of any soluble or membrane,His-tagged proteins. These large high-quality arrays are necessary for structural studies at high resolution. We have investigated the steps of adsorption and 2D crystallization of His-HupR using three different lipids: (i) 2-(bis-carboxymethyl-amino)-6-[2-(1,3-di-O-oleyl-glyceroxy)-acetyl-amino] hexanoic acid nickel-(II) (Ni-NTA-DOGA), which has been previously used, and two specifically designed Ni2+-chelating lipids, (ii) Ni-NTA-BB, which has two branched (B) alkyl chains and (iii) Ni-NTA-BF, a nonsymmetrical lipid with one branched (B) and one fluorinated (F) chain. These three lipids, when spread at the air-water interface, exhibit various fluidity properties. The adsorption and crystallization process have been monitored in situ band in real time using a variety of complementary techniques such as ellipsometry, shear rigidity measurements of the monolayer, and Brewster angle microscopy, and we have also developed X-ray :reflectivity analysis to investigate the evolution of the electron density profile of the lipid-protein monolayer. Electron microscopy observations of the protein-lipid layers were also performed. We have found that the fluidity of the lipid monolayer has a marked influence on the rates of protein adsorption and crystallization of His-HupR. When Ni-NTA-BB is used to form the monolayer, it accelerates the process of protein adsorption band the protein crystallization is three times faster than when Ni-NTA-DOGA is used.