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

• Published in: The Journal of biological chemistry Vol. 268; no. 6; pp. 3938 - 3943
• Main Authors: UTPAL BANIK, MANDAL, N. C, BHATTACHARYYA, B, SIDDHARTHA ROY
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-02-1993
• Subjects: Analytical, structural and metabolic biochemistry; Binding and carrier proteins; Biological and medical sciences; Biopolymers; Dansyl Compounds - chemistry; DNA-Binding Proteins; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Molecular Weight; phage lambda; Protein Conformation; Proteins; Repressor Proteins - chemistry; Thermodynamics; Transcription Factors - chemistry; Viral Proteins; Viral Regulatory and Accessory Proteins; Oligomerization; Structure activity relation; Gel electrophoresis; DNA binding protein; HPLC chromatography; Transcription repressor; Fluorescence spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)53562-6

Tetramer-dimer equilibrium of lambda repressor has been studied by fluorescence anisotropy techniques. We have chosen 1-dimethylamino
naphthalene-5-sulfonyl chloride (dansyl chloride)-labeled repressor to study the dissociation-association equilibrium, because
of relatively long life-time of the probe (> 10 ns). Polarization of the dansyl-labeled repressor decreases with decreasing
protein concentrations in the range of 20 to 0.2 microM. The decrease of anisotropy was shown to be due to reversible dissociation
of the protein. Size exclusion high-performance liquid chromatography studies and polyacrylamide gel electrophoresis under
native conditions (Ferguson plot) confirmed that at around 20 microM concentrations the repressor exists in predominantly
tetrameric form, whereas in lower concentrations it exists in predominantly dimer form. A dissociation constant of 2.3 +/-
0.9 microM was estimated in 0.1 M potassium phosphate, pH 8.0, at 25 degrees C. A stoichiometric amount of isolated single
operator shifted the tetramer-dimer equilibrium toward the dimer. Increased ionic strength had only a modest effect on the
dissociation constant. The thermodynamic constants for the dissociation reaction calculated from the Van't Hoff plot was +26.6
kcal/mol for delta H and +64.7 e.u. for delta S. The rotational correlation times derived from isothermal Perrin plot indicated
elongated dimers and tetramers.