Yu-Hung LinSung-Yao LinGuan-Syun LiShao-En WengShu-Ling TzengYu-Hsuan HsiaoNien-Jen Hu2026-01-302026-01-302019-09-1110.1038/s41598-019-49292-whttps://scholars.csmu.edu.tw/handle/123456789/3878<jats:title>Abstract</jats:title><jats:p>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<jats:sub>NM</jats:sub> 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.</jats:p>journal-article