Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.本研究围绕膜蛋白脂立方相结晶（meso crystallization）展开，旨在开发一种快速筛选技术：该技术整合了成熟经典气相扩散法（vapor diffusion experiment）与批量脂立方相结晶法（batch meso phase crystallization）的技术特点，且无需预先将蛋白与单油酰甘油（monoolein）混合。该技术兼具两种方法的核心优势：其一，可在脂立方相中稳定膜蛋白；其二，可通过气相扩散法精准调控水合水平与添加剂浓度；其三，大幅简化脂立方相结晶实验流程，且可兼容适配96孔板的标准液体处理机器人；其四，CIMP结晶法（CIMP crystallization）可直接监测相转变与结晶过程。利用该技术已获得高质量的嗜菌紫红质（Bacteriorhodopsin, BR）晶体，其衍射分辨率可达1.3 Å。本研究开发的CIMP结晶法及配套耗材与实验方案，已首次成功应用于获取盐沼盐杆菌（Halobacterium salinarum）来源的感光视紫红质II（sensory rhodopsin II, SRII）晶体。