Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins

• Published in: Scientific reports Vol. 9; no. 1; pp. 13171 - 9
• Main Authors: Lin, Yu-Hung, Lin, Sung-Yao, Li, Guan-Syun, Weng, Shao-En, Tzeng, Shu-Ling, Hsiao, Yu-Hsuan, Hu, Nien-Jen
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.09.2019; Nature Publishing Group
• Subjects: 631/1647/2258; 631/92/577; 82/29; 82/80; Alkylation; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Crystal structure; Cysteine; Cysteine - genetics; Cysteine - metabolism; Electrophoresis, Polyacrylamide Gel - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; Gel electrophoresis; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Membrane fusion; Membrane proteins; Models, Molecular; multidisciplinary; Mutation; Neisseria meningitidis - genetics; Neisseria meningitidis - metabolism; Organic Anion Transporters, Sodium-Dependent - chemistry; Organic Anion Transporters, Sodium-Dependent - genetics; Organic Anion Transporters, Sodium-Dependent - metabolism; Protein Conformation; Proteins; Reproducibility of Results; Science; Science (multidisciplinary); Sodium lauryl sulfate; Solvents; Solvents - chemistry; Symporters - chemistry; Symporters - genetics; Symporters - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-49292-w

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBT NM as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.