Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function.
The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U). The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.
Hobart
Sarah A
SA
Rensselaer Polytechnic Institute, Department of Chemistry, 110 8th Street, Troy, NY 12180, USA.
Ilin
Sergey
S
Moriarty
Daniel F
DF
Osuna
Robert
R
Colón
Wilfredo
W
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry* / Q000502:physiology; D005453:Fluorescence; D005454:Fluorescence Polarization; D007700:Kinetics; D011489:Protein Denaturation; D014443:Tyrosine / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D014443:Tyrosine; D014508:Urea