Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping.
Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping.
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DOI：
10.1007/978-1-62703-583-5_3
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Bowie JU
中科院分区：
文献类型：
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作者：
Hong H;Chang YC;Bowie JU
我们已经开发了一种方法来测量强跨膜（TM）的螺旋相互作用的亲和力，在脂双层，难以通过传统的稀释方法测量。该方法被称为空间捕获，将生物素化的TM螺旋的解离与单价链霉亲和素（mSA）的竞争性结合偶联，使得解离由mSA对生物素的亲和力和mSA浓度驱动。通过突变调节mSA的结合亲和力，该方法可以获得TM螺旋二聚体的解离常数（Kd，二聚体）在六个数量级的范围内。可测量的靶相互作用的Kd，二聚体极限比稀释方法可能的低3 - 4个数量级。因此，空间捕获开辟了新的机会，研究折叠和组装的α-螺旋膜蛋白在脂质双层环境。在这里，我们提供了将空间捕获应用于TM螺旋二聚体的详细方法。
We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (Kd,dimer) over a range of six orders of magnitudes. The Kd,dimer limit of measurable target interaction is extended 3–4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer.