Molecular recognition of histidine-tagged molecules by metal-chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy
Molecular recognition of histidine-tagged molecules by metal-chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy
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DOI：
10.1021/ja9735620
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发表时间：
1998-04-01
影响因子：
15
通讯作者：
Tampé, R
中科院分区：
文献类型：
--
作者：
Dorn, IT;Neumaier, KR;Tampé, R
蛋白质通过表面暴露的或蛋白质工程的组氨酸与金属螯合脂的复杂结合，为蛋白质在自组织界面上的定向和二维结晶提供了一个普遍而有力的概念。为了在分子水平上展示单个组氨酸标记分子和螯合剂脂质之间的配对形成，我们合成了同时含有镍-NTA螯合剂和荧光基团的新型脂类。这些脂质作为光谱探针，利用荧光共振能量转移(FRET)直接观察金属螯合脂质对荧光标记的组氨酸标记多肽的分子识别。分子对接到组装在单层或双层中的螯合脂质具有很高的特异性，只有3%的非特异性吸附和结合常数为3µM。亲和力常数通过单分子的荧光相关光谱(FCS)证实，其中脂质结合/游离的比率是通过大约1fl的共焦体积的本质不同的扩散时间来分析的。通过使用模型肽，可以忽略大部分静电和空间位阻对结合过程的贡献。因此，亲和常数可以作为组氨酸标记的蛋白质与螯合剂脂质界面结合的标准值。
Complex binding of proteins by metal-chelating Lipids via surface-exposed or protein-engineered histidines provides an universal and powerful concept for the orientation and two-dimensional crystallization of proteins at self-organized interfaces. To demonstrate pair formation between individual histidine-tagged molecules and chelator lipids on the molecular level, we have synthesized novel lipids bearing both a Ni-NTA chelator and a fluorescent group. These lipids serve as spectroscopic probes to visualize directly the molecular recognition of fluorescence-labeled histidine-tagged peptides by metal-chelating lipids using fluorescence resonance energy transfer (FRET). The molecular docking to chelator lipids assembled in mono- or bilayers is highly specific, revealing only 3% unspecific adsorption and a binding constant of 3 mu M. The affinity constant was confirmed by fluorescence correlation spectroscopy (FCS) on single molecules, where the ratio of lipid-bound to free was analyzed by their intrinsically different diffusion times passing through a confocal volume of about 1 fL. By using a model peptide most of the electrostatic and steric contribution to the binding process can be neglected. Therefore, the affinity constant can serve as a standard value for the binding of histidine-tagged proteins to chelator lipid interfaces.