A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane‐mimetics using GB1 fusion proteins and NMR spectroscopy
A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane‐mimetics using GB1 fusion proteins and NMR spectroscopy
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DOI：
10.1002/pro.2127
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发表时间：
2012-10
期刊：
影响因子：
8
通讯作者：
Lisa A. M. Sommer;M. Meier;S. Dames
中科院分区：
文献类型：
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作者：
Lisa A. M. Sommer;M. Meier;S. Dames
链球菌蛋白G(GB1)的B1区融合多肽和蛋白质的表达非常受欢迎，因为GB1通常可以改善目的蛋白的溶解性，而且利用免疫球蛋白亲和层析纯化的第一步简单有效。然而，下面的蛋白酶消化并不总是完整的，或者可能导致目标蛋白质的消化。此外，还需要进一步的纯化步骤，如反相高效液相色谱法，以去除GB1标签和未消化的融合蛋白。由于蛋白酶的消化和后续的纯化步骤不仅耗时而且通常也很昂贵，我们测试了GB1融合蛋白是否可以直接用于类脂或膜仿制的核磁共振相互作用研究。基于仅使用GB1部分的核磁共振结合研究，该融合标签与不同的膜模拟物如胶束、双胶束或脂质体没有显著的相互作用。因此，使用GB1融合蛋白观察到的光谱变化表明目标蛋白的脂膜相互作用。该方法最初的建立是为了探测Ser/Thr激酶TOR的FATC结构域的大量突变体的膜相互作用。为了证明该方法的有效性，我们展示了野生型蛋白和一个亮氨酸与丙氨酸突变体的核磁共振结合数据。
The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP‐HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time‐consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane‐mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane‐mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1‐fusion proteins indicate lipid‐ and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.