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A fluorescence anisotropy study of tetramer-dimer equilibrium of lambda repressor and its implication for function

U Banik1, N C Mandal, B Bhattacharyya

  • 1Department of Biophysics, Bose Institute, Calcutta, India.

The Journal of Biological Chemistry
|February 25, 1993
PubMed
Summary

Lambda repressor undergoes tetramer-dimer dissociation, shifting towards dimers at lower concentrations. This equilibrium is influenced by operator binding and ionic strength, providing insights into protein assembly.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Dynamics

Background:

  • The lambda repressor protein plays a crucial role in bacteriophage lambda DNA replication and lysogeny.
  • Understanding the oligomerization state of lambda repressor is key to elucidating its regulatory mechanisms.
  • Protein concentration-dependent equilibria, such as tetramer-dimer transitions, are common in transcriptional regulators.

Purpose of the Study:

  • To investigate the tetramer-dimer equilibrium of lambda repressor using biophysical techniques.
  • To quantify the dissociation constant and thermodynamic parameters governing this equilibrium.
  • To assess the influence of operator binding and ionic strength on the repressor's oligomeric state.

Main Methods:

  • Fluorescence anisotropy using a dansyl chloride-labeled lambda repressor.

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  • Size exclusion high-performance liquid chromatography (SE-HPLC).
  • Native polyacrylamide gel electrophoresis (native PAGE) with Ferguson plot analysis.
  • Van't Hoff and Perrin plot analyses for thermodynamic and rotational dynamics.

    • Main Results:

    • Fluorescence anisotropy decreased with decreasing protein concentration (0.2–20 microM), indicating reversible dissociation.
    • SE-HPLC and native PAGE confirmed a shift from tetrameric to dimeric forms at lower concentrations.
    • A dissociation constant (Kd) of 2.3 ± 0.9 microM was determined at 0.1 M potassium phosphate, pH 8.0, 25°C.
    • Operator binding favored the dimeric state, while increased ionic strength had a minor effect on Kd.
    • Thermodynamic analysis yielded ΔH = +26.6 kcal/mol and ΔS = +64.7 e.u.
    • Rotational correlation times suggested elongated dimer and tetramer structures.

    Conclusions:

    • Lambda repressor exists in a concentration-dependent tetramer-dimer equilibrium, with dimers predominating at lower concentrations.
    • The dissociation process is reversible and influenced by operator binding, suggesting a mechanism for gene regulation.
    • The thermodynamic and structural data provide fundamental insights into the assembly and function of lambda repressor.