Hong Heedeok, Chang Yu-Chu, Bowie James U
Department of Chemistry, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2013;1063:37-56. doi: 10.1007/978-1-62703-583-5_3.
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 (K d,dimer) over a range of six orders of magnitudes. The K d,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.
我们开发了一种方法来测量脂质双层中强跨膜（TM）螺旋相互作用亲和力，而这是传统稀释方法难以测量的。这种方法称为空间捕获，它将生物素化的TM螺旋的解离与单价链霉亲和素（mSA）的竞争性结合相结合，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，该方法可以在六个数量级的范围内获得TM螺旋二聚体的解离常数（Kd，二聚体）。可测量的目标相互作用的Kd，二聚体极限比稀释方法所能达到的低3 - 4个数量级。因此，空间捕获为研究脂质双层环境中α - 螺旋膜蛋白的折叠和组装开辟了新机会。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。
