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Updated: Feb 12, 2026
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Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers
Published on: March 21, 2025
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Oversized galactosides as a probe for conformational dynamics in LacY
Irina Smirnova1, Vladimir Kasho1, Xiaoxu Jiang1
1Department of Physiology, University of California, Los Angeles, CA 90095.
Summary
The lactose permease (LacY) in E. coli binds large galactoside derivatives, showing its binding site is accessible even for bulky ligands. This suggests a wider entry pathway than previously thought.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The lactose permease (LacY) from Escherichia coli is a model transporter for studying sugar uptake mechanisms.
- Understanding LacY's substrate binding and transport pathway is crucial for elucidating membrane protein function.
Purpose of the Study:
- To investigate the binding kinetics of alpha-galactopyranoside homologs with varying aglycone sizes and shapes to E. coli's LacY.
- To determine the accessibility of the LacY binding site and the nature of the substrate entry pathway.
Main Methods:
- Förster resonance energy transfer (FRET) was used to measure binding kinetics.
- Mutagenesis studies, including replacing Phe27 with Tryptophan, were performed.
- Binding kinetics were analyzed for wild-type LacY, LacY/nanobody complexes, and mutant variants.
Main Results:
- Fast binding kinetics (k_on = 4-20 μM⁻¹·s⁻¹) were observed for LacY in an outward-open conformation, even with ligands larger than lactose.
- Dissociation rates (k_off) increased with aglycone size, but dissociation constants (K_d) remained in the micromolar range.
- Mutating Phe27 did not impede binding of large ligands, and the periplasmic opening was found to be wider than suggested by X-ray structures.
Conclusions:
- E. coli's LacY exhibits high accessibility to its binding site, accommodating large galactoside derivatives.
- The substrate entry pathway on the periplasmic side is wider than previously estimated from structural data.
- LacY specificity is primarily determined by the galactopyranoside ring, with flexibility in accommodating diverse aglycones.
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