Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy.
High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.
Royer
C A
CA
Department of Physics, University of Illinois, Urbana 61801.
Chakerian
A E
AE
Matthews
K S
KS
D003619:Dansyl Compounds; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007700:Kinetics; D046911:Macromolecular Substances; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*
D003619:Dansyl Compounds; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors