Zhang Huamin, Cramer William A
Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN 47907-2054, USA.
J Struct Funct Genomics. 2005;6(2-3):219-23. doi: 10.1007/s10969-005-1912-y.
The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein-protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b(6)f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein-protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein-protein contacts that facilitated crystallization.
导致完整膜蛋白结构解析进展缓慢的主要障碍是将去污剂溶解的疏水蛋白，尤其是异源寡聚完整膜蛋白结晶的困难。对于后一类多亚基蛋白，除了普遍存在的去污剂兼容性问题外，我们还遇到了以下问题：（i）过度纯化导致的不稳定性，进而引起脱脂；（ii）蛋白酶活性降解复合物亚基暴露的环和末端，且无法抑制；（iii）蛋白质-蛋白质接触不佳，可能是由于去污剂胶束的掩盖所致。问题（i）可通过用合成脂质增强脱脂复合物，在细胞色素b（6）f复合物的结晶过程中得到改善。问题（ii）尚未解决。问题（iii）在其他系统中已通过使用单克隆抗体（或其他蛋白质配体）来增加蛋白质-蛋白质接触的概率得以解决。在钴胺素与大肠杆菌素受体BtuB以及大肠杆菌素E3的受体结合域形成的复合物中，后者作为促进结晶的蛋白质-蛋白质接触的配体。
