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Inducible Operons: lac Operon01:25

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The lac operon in Escherichia coli is a model for understanding inducible gene regulation and metabolic flexibility. It integrates local control by lactose and global regulation through catabolite repression, enabling E. coli to preferentially metabolize glucose when available and switch to lactose utilization when glucose is scarce.Structure and Function of the lac OperonThe lac operon contains three structural genes: lacZ (β-galactosidase), lacY (lactose permease), and lacA...
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Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
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Real-time conformational changes in LacY.

Irina Smirnova1, Vladimir Kasho1, H Ronald Kaback2

  • 1Departments of Physiology and.

Proceedings of the National Academy of Sciences of the United States of America
|May 30, 2014
PubMed
Summary

Lactose permease (LacY) in E. coli uses an alternating access mechanism. Galactoside binding controls LacY conformational changes, with periplasmic cavity opening limiting substrate access and cytoplasmic cavity closing.

Keywords:
major facilitator superfamilymembrane transport proteins

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Area of Science:

  • Membrane protein structure and function
  • Biochemistry and biophysics
  • Molecular transport mechanisms

Background:

  • Lactose permease (LacY) facilitates galactoside/H(+) symport in E. coli via an alternating access mechanism.
  • This mechanism involves sequential opening and closing of cavities on the periplasmic and cytoplasmic sides of the protein.

Purpose of the Study:

  • To investigate the real-time conformational changes in LacY upon galactoside binding.
  • To determine the rates of cavity opening and closing in LacY.
  • To elucidate how these conformational changes influence substrate binding and transport.

Main Methods:

  • Utilized tryptophan quenching/unquenching of bimane, a covalently attached fluorophore, to monitor LacY conformational dynamics.
  • Employed stopped-flow techniques to measure bimane fluorescence changes in real-time.
  • Assayed LacY in both detergent solution and proteoliposomes to compare functional states.

Main Results:

  • Galactoside binding initiates real-time conformational changes in LacY.
  • The rate of periplasmic cavity opening, measured by bimane unquenching, is 20-30 s⁻¹ and independent of sugar concentration.
  • Periplasmic cavity opening limits substrate access and controls the rate of cytoplasmic cavity closing, which is also sugar-independent.

Conclusions:

  • Periplasmic cavity opening is a rate-limiting step for substrate binding to LacY.
  • The opening of the periplasmic cavity dictates the rate of cytoplasmic cavity closure, coordinating the alternating access cycle.
  • These findings provide insights into the dynamic mechanism of secondary active transport mediated by LacY.