Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
J Struct Biol. 2010 Jul;171(1):82-7. doi: 10.1016/j.jsb.2010.02.005. Epub 2010 Feb 10.
Membrane protein crystallography frequently stalls at the phase determination stage due to poor crystal diffraction and the inability to identify heavy atom derivatization prior to data collection. Thus, a majority of time, effort and resources are invested preparing potential derivatized crystals for synchrotron data collection and analysis without knowledge of heavy atom labeling. To remove this uncertainty, we introduce Fluorescence Detection of Heavy Atom Labeling (FD-HAL) using tetramethylrhodamine-5-maleimide (a fluorescent maleimide compound) to monitor in-gel cysteine residue accessibility and ascertain covalent modification by mercury, platinum and gold compounds. We have tested this technique on three integral membrane proteins (LacY, vSGLT and mVDAC1) and can quickly assess the optimal concentrations, time and heavy atom compound to derivatize free cysteine residues in order to facilitate crystal phasing. This, in conjunction with cysteine scanning for incorporating heavy atoms at strategic positions, is a useful tool that will considerably assist in phasing membrane protein structures.
膜蛋白晶体学经常在相位确定阶段停滞不前，原因是晶体衍射较差，并且在收集数据之前无法识别重原子衍生化。因此，大多数时间、精力和资源都投入到准备潜在的衍生化晶体进行同步辐射数据收集和分析上，而不知道重原子标记。为了消除这种不确定性，我们引入了使用四甲基罗丹明-5-马来酰亚胺（一种荧光马来酰亚胺化合物）的重原子标记荧光检测（FD-HAL），以监测胶内半胱氨酸残基的可及性，并确定汞、铂和金化合物的共价修饰。我们已经在三种整合膜蛋白（LacY、vSGLT 和 mVDAC1）上测试了该技术，并且可以快速评估最佳浓度、时间和重原子化合物，以衍生游离半胱氨酸残基，从而促进晶体相位测定。这与在战略位置掺入重原子的半胱氨酸扫描相结合，是一种非常有用的工具，将极大地帮助膜蛋白结构的相位测定。
