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.
Highlights
In the past decades, a number of methodologies have been developed for experimental validation of predicted topology maps
It has been shown that mPEG-MAL-5K can label a solvent accessible cysteine residues of an inner membrane protein on intact E. coli cell membrane[19]
The PEGylation results showed that cfASBTNM A275C was chemically modified with mPEGMAL-5K as the fluorescent protein band shifted approximately 5 kDa (Figs 1c, 2a and Supplementary Fig. S1b), indicating mPEG-MAL-5K can permeate the outer membrane of E. coli
Summary
A number of methodologies have been developed for experimental validation of predicted topology maps (for detailed overviews see, for example[3,4]). A common strategy is to construct a chimera fusion with reporter enzymes attached to the terminus of C-terminal truncated target membrane proteins, such as alkaline phosphatase PhoA5, β-galactosidase LacZ6, and β-lactamase bla[7]. The sulfhydryl-reactive reagent, methoxypolyethylene glycol maleimide 5 K (mPEG-MAL-5K), covalently attaches to solvent accessible cysteine residues on the surface of intact cells, resulting in a 5 kDa band shift observable by in-gel fluorescence. The PEGylated levels of cfASBTNM A275C-EGFP were calculated according to Eq (1), and plotted against different protein to mPEG-MAL-5K molar ratios. (c,d) PEGylation of cfASBTNM D61C-EGFP fusion as monitored by in-gel fluorescence using permeabilized membranes treated with mPEG-MAL-5K. Lanes 3–7: Permeabilized membranes containing cfASBTNM D61C-EGFP treated with indicated protein to mPEG-MAL-5K molar ratios. An efficient and direct method for topological mapping and characterization of the conformational variations of membrane proteins in native membrane environment
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