Correlating membrane-protein dynamics with function: Integrating bioinformatics, molecular dynamics, and...

Area of Science:

• Structural biology
• Biophysics
• Biochemistry

Background:

• Integral membrane proteins play vital roles in cellular processes.
• Understanding their conformational dynamics is key to elucidating function.
• The small monotopic phosphoglycosyl transferase (SmPGT) superfamily is essential for glycoconjugate biosynthesis in prokaryotes.

Purpose of the Study:

• To develop and apply an integrated strategy for observing ligand-dependent conformational dynamics of integral membrane proteins in situ.
• To investigate the structure-function relationship within the SmPGT superfamily.
• To validate the role of protein motion in ligand binding for PglC.

Main Methods:

• Integrated approach using structural bioinformatics, molecular simulation, and single-molecule Förster Resonance Energy Transfer (FRET) microscopy.
• Development of a platform for monitoring intramolecular protein dynamics in a native-like lipid environment using styrene-maleic acid liponanoparticles (SMALPs).
• Utilized selective cysteine protein labeling, non-canonical amino acid mutagenesis, and click chemistry to create dual-labeled PglC variants.

Main Results:

• Identified substrate-specific structural features across the SmPGT superfamily.
• Correlated ligand-dependent conformational dynamics with structural features using all-atom simulations.
• Demonstrated that conformational changes of PglC upon inhibitor binding correlate with inhibitor potency.

Conclusions:

• The developed single-molecule FRET-SMALP strategy effectively monitors protein dynamics in a native-like membrane environment.
• This approach is adaptable for studying diverse SmPGTs with varying substrate specificities.
• Structure prediction and molecular dynamics support significant conformational changes upon ligand binding in this protein superfamily.