Lee J W, Chan M, Law T V, Kwon H J, Jap B K
Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
J Mol Biol. 1995 Sep 8;252(1):15-9. doi: 10.1006/jmbi.1994.0470.
Ubiquinol-cytochrome c reductase is a crucial integral membrane protein in the mitochondrial respiratory cycle. Eleven subunits containing three cytochrome heme groups and a 2Fe-2S Rieske center make up this 240 kDa enzyme complex. Previously, many different crystal forms of the bc1 complex have displayed diffraction to as far as 4.5 A. However, rapid degradation of the protein in the X-ray beam at room temperature has obstructed the collection of a full data set from a single crystal. As slight heterogeneities between crystals severely hampered merging of data from different crystals, we sought a method to stabilize the protein crystal in the X-ray beam in order to collect a full data set from one crystal sample. To this end, water soluble protein crystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In this communication, we report on a successful experiment in which flash-cooled bc1 membrane protein crystals have given rise to sustained diffraction over a 60 hour data collection period at a synchrotron source. Furthermore, we present an improved purification and crystallization protocol yielding crystals readily diffracting out to 3.3 A. These results should greatly aid in the future realization of the molecular structure of the bc1 complex as well as other membrane proteins.
泛醇 - 细胞色素c还原酶是线粒体呼吸循环中一种关键的整合膜蛋白。这个240 kDa的酶复合物由包含三个细胞色素血红素基团和一个2Fe - 2S 里氏中心的11个亚基组成。此前，bc1复合物的许多不同晶体形式已显示出高达4.5 Å的衍射。然而，在室温下蛋白质在X射线束中快速降解阻碍了从单个晶体收集完整数据集。由于晶体之间的轻微异质性严重妨碍了来自不同晶体的数据合并，我们寻求一种方法来稳定蛋白质晶体在X射线束中的状态，以便从一个晶体样品收集完整数据集。为此，水溶性蛋白质晶体经常被快速冷却到低温；然而，尚无关于膜蛋白低温晶体学的报道。在本通讯中，我们报告了一个成功的实验，其中快速冷却的bc1膜蛋白晶体在同步辐射源60小时的数据收集期间产生了持续的衍射。此外，我们提出了一种改进的纯化和结晶方案，得到的晶体易于衍射至3.3 Å。这些结果应极大地有助于未来实现bc1复合物以及其他膜蛋白的分子结构。
