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. Ligand binding increases the rate of PEGylation at five out of eight tested positions on the periplasmic side in vivo, while decreasing the rate of PEGylation at both positions tested on the cytoplasmic side in situ. Upon ligand binding, the rate of PEGylation at two periplasmic positions, K42 and Q242, slightly decreases in vivo, but increases in situ, indicating the conformational behavior of these two residues in living cells may not be identical to that in isolated cell membranes. Furthermore, abolishing the electrochemical H+ gradient (Δμ∼H+) reduces the rate of PEGylation at all tested positions on the periplasmic side. We also found that, unlike the linear form, the branched (Y-shape) mPEG-Mal-5K cannot pass the outer membrane. This work characterizes the alternating access of LacY in the context of a living cell and demonstrates that this methodology is feasible and effective for dynamical studies of MFS transporters.