Musial-Siwek Monika, Jaffee Marcie B, Imperiali Barbara
Departments of Biology and Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
J Am Chem Soc. 2016 Mar 23;138(11):3806-12. doi: 10.1021/jacs.5b13426. Epub 2016 Mar 14.
Integral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally determined and computationally predicted structures are urgently needed. Herein we present the application of luminescence resonance energy transfer (LRET) for investigating the interactions of the polytopic membrane-bound oligosaccharyl transferases (OTases) with partner substrates. Monomeric OTases, such as the PglBs from Campylobacter jejuni and Campylobacter lari, catalyze transfer of glycans from membrane-associated undecaprenol diphosphate-linked substrates to proteins in the bacterial periplasm. LRET-based distance measurements are enabled by the inclusion of an encoded N-terminal lanthanide-binding tag (LBT), and LRET between the luminescent (LBT)-Tb(3+) donor complex and fluorescently labeled peptide and glycan substrates provides discrete distance measurements across the span of the membrane. LRET-based measurements of detergent-solubilized PglB from C. lari allowed direct comparison with the distances based on the previously reported the C. lari PglB crystal structure, thereby validating the approach in a defined system. Distance measurements between peptide and glycan substrates and the C. jejuni PglB offer new experimental information on substrate binding to the related, but structurally uncharacterized, eukaryotic OTase.
整合膜蛋白在所有生命系统中都发挥着至关重要的作用；然而，这类蛋白的分析面临着重大技术障碍。迫切需要生物物理方法来提供结构信息，以补充和利用实验测定及计算预测的结构。在此，我们展示了发光共振能量转移（LRET）在研究多跨膜结合寡糖基转移酶（OTases）与伴侣底物相互作用中的应用。单体OTases，如空肠弯曲菌和海鸥弯曲菌的PglBs，催化聚糖从膜相关的十一异戊二烯二磷酸连接的底物转移至细菌周质中的蛋白质。基于LRET的距离测量通过包含编码的N端镧系元素结合标签（LBT）来实现，并且发光（LBT）-Tb(3+)供体复合物与荧光标记的肽和聚糖底物之间的LRET提供了跨膜跨度的离散距离测量。基于LRET对来自海鸥弯曲菌的去污剂溶解的PglB的测量，使得能够与基于先前报道的海鸥弯曲菌PglB晶体结构的距离进行直接比较，从而在一个确定的系统中验证了该方法。肽和聚糖底物与空肠弯曲菌PglB之间的距离测量为底物与相关但结构未表征的真核OTase的结合提供了新的实验信息。
