Probing Ligand-Induced Conformational Changes in an MFS Transporter in vivo Using Site-Directed PEGylation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Site-directed alkylation (SDA) studies on Major Facilitator Superfamily (MFS) transporters often lack native cellular conditions.
• Understanding transporter dynamics in vivo is crucial for elucidating their mechanisms.

Purpose of the Study:

• To investigate ligand-induced conformational changes of the lactose permease (LacY) in Escherichia coli within a living cell environment.
• To establish and validate a GFP-based site-directed PEGylation method for in vivo studies of MFS transporters.

Main Methods:

• Utilized a Cys-less LacY-eGFP fusion protein with single-Cys replacements.
• Employed methoxy polyethylene glycol-maleimide-5K (mPEG-Mal-5K) for site-directed PEGylation.
• Analyzed PEGylation-induced band shifts via in-gel fluorescence under various conditions (ligand presence/absence, electrochemical gradient).

Main Results:

• Ligand binding accelerated periplasmic PEGylation at five sites and decelerated cytoplasmic PEGylation at two sites in vivo.
• Conformational differences were observed for residues K42 and Q242 between in vivo and in situ conditions.
• Abolishing the H+ gradient reduced periplasmic PEGylation rates.
• Branched mPEG-Mal-5K showed limited ability to cross the outer membrane.

Conclusions:

• Demonstrated the feasibility of in vivo site-directed PEGylation for studying MFS transporter dynamics.
• Characterized the alternating access mechanism of LacY in a native cellular context.
• Highlighted the importance of cellular environment on transporter conformation and function.