A fluorescence anisotropy study of tetramer-dimer equilibrium of lambda repressor and its implication for function.
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.
Banik
U
U
Department of Biophysics, Bose Institute, Calcutta, India.
Mandal
N C
NC
Bhattacharyya
B
B
Roy
S
S
D001704:Biopolymers; D004268:DNA-Binding Proteins; D003619:Dansyl Compounds / Q000737:chemistry; D005454:Fluorescence Polarization; D008970:Molecular Weight; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001704:Biopolymers; D004268:DNA-Binding Proteins; D003619:Dansyl Compounds; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C029038:dansyl chloride