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Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins.
- Source :
-
Scientific Reports . 9/11/2019, Vol. 9 Issue 1, pN.PAG-N.PAG. 1p. - Publication Year :
- 2019
-
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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 9
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Scientific Reports
- Publication Type :
- Academic Journal
- Accession number :
- 138577931
- Full Text :
- https://doi.org/10.1038/s41598-019-49292-w