Probing ligand-induced conformational changes in an MFS transporter in vivo using site-directed PEGylation.

So far, site-directed alkylation (SDA) studies on transporters in the Major Facilitator Superfamily (MFS) are mostly performed at conditions different from the native cellular environment. In this study, using GFP-based site-directed PEGylation, ligand-induced conformational changes in the lactose permease of Escherichia coli (LacY), were examined in vivo for the first time. Accessibility/reactivity of single-Cys replacements in a Cys-less LacY-eGFP fusion background was tested using methoxy polyethylene glycol-maleimide-5K (mPEG-Mal-5K) in the absence or presence of a ligand, and the band-shift of the fusion upon PEGylation was detected by in-gel fluorescence.