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Site-directed alkylation and the alternating access model for LacY
H Ronald Kaback1, R Dunten, S Frillingos
1Department of Physiology, University of California, Los Angeles, CA 90095-1662, USA. rkaback@mednet.ucla.edu
Summary
Researchers studied the lactose permease (LacY) in E. coli by replacing residues with cysteine. Ligand binding alters cysteine reactivity, supporting a model of alternating access for sugar transport.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Transport
Background:
- The lactose permease (LacY) from Escherichia coli is a model transporter.
- Understanding LacY's mechanism is crucial for membrane transport research.
- Previous studies have focused on structural and functional aspects of LacY.
Purpose of the Study:
- To investigate the structural basis of LacY function by probing residue accessibility.
- To map solvent accessibility and ligand-induced conformational changes in LacY.
- To test a model of alternating access mechanism in LacY.
Main Methods:
- Site-directed mutagenesis to introduce cysteine residues into a cysteine-free LacY mutant.
- Reactivity assays using N-ethylmaleimide (NEM) in right-side-out membrane vesicles.
- Analysis of reactivity in the presence and absence of lactose ligand.
Main Results:
- Engineered cysteines in solvent-accessible regions and the inward-facing cavity showed high NEM reactivity.
- Cysteines in membrane-embedded or tightly packed regions exhibited low reactivity.
- Lactose binding increased reactivity of periplasmic-side cysteines and decreased reactivity of cytoplasmic-side cysteines.
- Reactivity changes were observed at opposite ends of helices, suggesting cavity dynamics.
Conclusions:
- The results support an alternating access model for LacY.
- Conformational changes involving periplasmic and cytoplasmic cavities mediate substrate transport.
- LacY's mechanism involves dynamic opening and closing of internal cavities.
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