Lin Sung-Yao, Sun Xing-Han, Hsiao Yu-Hsuan, Chang Shao-En, Li Guan-Syun, Hu Nien-Jen
Institute of Biochemistry, National Chung Hsing University, Taichung, 40227, Taiwan, ROC.
PLoS One. 2016 Jun 22;11(6):e0157923. doi: 10.1371/journal.pone.0157923. eCollection 2016.
Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.
膜蛋白在细胞的许多基本功能中发挥着关键作用，包括ATP合成、离子和分子转运、细胞信号传导以及酶促反应，约占整个基因组基因的30%。然而，膜蛋白的疏水性常常阻碍结构测定的进展。去污剂筛选是获得稳定的去污剂增溶膜蛋白和良好衍射蛋白晶体的关键步骤。荧光检测尺寸排阻色谱法（FSEC）已被开发用于监测膜蛋白在去污剂胶束中的提取效率和单分散性。通过追踪绿色荧光蛋白（GFP）融合膜蛋白的FSEC图谱，该方法显著提高了去污剂筛选的通量。然而，目前获取FSEC图谱的方法要么需要配备在线荧光检测器的尺寸排阻色谱（SEC）设备，要么需要离线的荧光分光光度计微孔板读数器。在此，我们介绍一种替代方法，通过检测GFP在485nm处的吸光度（FA-SEC），从而通过在线吸光光谱仪在传统的SEC设备上实现该方法。结果表明，吸光度与GFP的荧光高度相关。通过使用更长光程的流通池，可以改善相对较弱的吸收信号。FA-SEC图谱与FSEC绘制的图谱一致，表明FA-SEC可能是用于膜蛋白去污剂筛选的一种可比且经济的设置。
