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

doi:10.1038/s41598-019-49292-w

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學系 > 期刊論文 > Item 310902500/20456

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/20456

Title:	Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins
Authors:	Lin, Y.-H. Lin, S.-Y. Li, G.-S. Weng, S.-E. Tzeng, S.-L. Hsiao, Y.-H. Hu, N.-J.
Contributors:	中山醫學大學醫學系
Keywords:	Symporters;Membrane Transport Proteins;Lactose permease
Date:	2019-12
Issue Date:	2019-11-21T02:42:53Z (UTC)
Publisher:	Scientific Reports
ISSN:	2045-2322
Abstract:	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 ASBTNM 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. © 2019, The Author(s).
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/20456
Relation:	Scientific Reports, Volume 9, Issue 1, NO. 13171
Appears in Collections:	[醫學系] 期刊論文

Files in This Item:

File	Description	Size	Format
SiteDirected-Alkylation-Detected-by-InGel-Fluorescence-SDAF-to-Determine-the-Topology-Map-and-Probe-the-Solvent-Accessibility-of-Membrane-Proteins2019Scientific-Reports.pdf		2153Kb	Adobe PDF	176	View/Open

View Licence

Loading...