Cleveland Iv Thomas, Blick Emily, Krueger Susan, Leung Anna, Darwish Tamim, Butler Paul
National Institute of Standards and Technology and Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
National Institute of Standards and Technology Center for Neutron Research, 100 Bureau Drive, Gaithersburg, MD 20899, USA.
IUCrJ. 2021 Jan 1;8(Pt 1):22-32. doi: 10.1107/S2052252520013974.
Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubilizing detergent. A better fundamental understanding of the behavior of detergents in the LCP may guide and simplify the detergent selection process. This work investigates the distribution of protein and detergent in LCP using the membrane protein bacteriorhodopsin (bR), with the LCP prepared from highly deuterated monoolein to allow contrast-matched small-angle neutron scattering. Contrast-matching allows the scattering from the LCP bilayer itself to be suppressed, so that the distribution and behavior of the protein and detergent can be directly studied. The results showed that, for several common detergents, the detergent micelle dissociates and incorporates into the LCP bilayer essentially as free detergent monomers. In addition, the detergent octyl glucoside dissociates from bR, and neither the protein nor detergent forms clusters in the LCP. The lack of detergent assemblies in the LCP implies that, upon incorporation, micelle sizes and protein/detergent interactions become less important than they would be in solution crystallization. Crystallization screening confirmed this idea, with crystals obtained from bR in the presence of most detergents tested. Thus, in LCP crystallization, detergents can be selected primarily on the basis of protein stabilization in solution, with crystallization suitability a lesser consideration.
脂质立方相（LCP）结晶方法对于获得某些膜蛋白的晶体至关重要，尤其是G蛋白偶联受体。LCP结晶通常需要对大量潜在变量进行优化，其中之一可能是增溶去污剂的选择。对去污剂在LCP中的行为有更深入的基本了解可能会指导并简化去污剂的选择过程。这项工作使用膜蛋白细菌视紫红质（bR）研究了LCP中蛋白质和去污剂的分布，所制备的LCP由高度氘代的单油酸甘油酯制成，以便进行对比度匹配的小角中子散射。对比度匹配可抑制来自LCP双层本身的散射，从而可以直接研究蛋白质和去污剂的分布及行为。结果表明，对于几种常见的去污剂，去污剂胶束会解离并基本上以游离去污剂单体的形式掺入LCP双层中。此外，去污剂辛基葡糖苷会从bR上解离，蛋白质和去污剂在LCP中均不会形成聚集体。LCP中不存在去污剂聚集体意味着，在掺入后，胶束大小和蛋白质/去污剂相互作用的重要性低于它们在溶液结晶中的重要性。结晶筛选证实了这一观点，在大多数测试的去污剂存在下，从bR中获得了晶体。因此，在LCP结晶中，可以主要根据蛋白质在溶液中的稳定性来选择去污剂，而较少考虑结晶适用性。
