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Related Experiment Videos

The lactose permease meets Frankenstein

H R Kaback1, S Frillingos, H Jung

  • 1Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles 90024-1662.

The Journal of Experimental Biology
|November 1, 1994
PubMed
Summary

The lactose permease in E. coli, a key membrane transporter, facilitates the coupled movement of beta-galactosides and H+ across cell membranes. Site-directed fluorescence spectroscopy is used to investigate its structure-function relationships.

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Area of Science:

  • Membrane biology
  • Protein structure and function
  • Biochemistry

Background:

  • The lactose permease (lacY) of Escherichia coli is a model system for understanding membrane transport proteins.
  • It catalyzes the symport of beta-galactosides and protons (H+) with a 1:1 stoichiometry.
  • Previous studies suggested a secondary structure model with 12 transmembrane alpha-helical domains.

Purpose of the Study:

  • To investigate the structure-function relationships of the lactose permease.
  • To confirm the proposed secondary structure and topology of the lactose permease.
  • To utilize site-directed fluorescence spectroscopy for detailed structural analysis.

Main Methods:

  • Solubilization, purification, and reconstitution of lactose permease into phospholipid vesicles.

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  • Circular dichroism and other spectroscopic techniques to assess secondary structure.
  • Hydropathy analysis of the amino acid sequence and studies using lac permease/alkaline phosphatase fusion proteins.
  • Site-directed fluorescence spectroscopy.

    • Main Results:

    • The purified lactose permease is approximately 80% helical, consistent with alpha-helical transmembrane domains.
    • Hydropathy analysis and fusion protein studies support a 12-helix transmembrane model with N and C termini on the inner membrane surface.
    • Site-directed fluorescence spectroscopy provides insights into structure-function relationships.

    Conclusions:

    • The 12-helix model for lactose permease structure is well-supported by multiple experimental approaches.
    • Lactose permease functions as a proton symporter with a defined stoichiometry.
    • Site-directed fluorescence spectroscopy is a valuable tool for probing membrane protein structure and dynamics.