Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function

Abstract

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.

Fig. 1.

A ribbon diagram of the 3D structure of FIS (Safo et al. 1997) showing the tyrosine residues. The four α-helices are labeled A through D. The program MOLSCRIPT (Kraulis 1991) was used to draw the figure.

Fig. 2.

Urea-induced denaturation of FIS monitored by (A) far-UV CD at 222 nm, and (B) near-UV CD at 277 nm. FIS concentrations were 107 μM (○), 35.6 μM (♦), 8.9 μM (□), or 1.8 μM (•). The insets in (A) and (B) show the full wavelength scans in the far- and near-UV regions, respectively. The molar ellipticity, [θ], has units of degree•cm²•dmol⁻¹. The symbols in inset (A) represent the full spectrum of native (○), unfolded in 6 M urea (+), and refolded (×) FIS. Error bars represent the average of at least three individual data sets. The solid lines on the denaturation curve indicate the fit for each data set as described in Materials and Methods.

Fig. 3.

Urea-induced denaturation of FIS monitored by (A) fluorescence and (B) steady-state anisotropy at 107 μM (○), 35.6 μM (♦), or 8.9 μM (□) FIS. The excitation wavelength was 276 nm, and the emission wavelength was 305 nm. Error bars represent the average of at least three individual data sets. The solid lines indicate the fit for each data set as described in Materials and Methods.

Fig. 4.

An overlay of the urea denaturation curves of FIS determined by far-UV CD (•), near-UV CD (□), fluorescence (⋄), and anisotropy (+) at FIS concentrations of 8.9 μM (A), 35.6 μM (B), or 107 μM (C). The data was normalized to apparent fraction unfolded using equation 4.

Fig. 5.

The change in signal upon FIS dilution as monitored by (A) CD and (B) size-exclusion HPLC. The solid line in (A) indicates the fit of the data as described in Materials and Methods. The chromatographic traces (B) show a representative elution profile at 29, 22, 9.9, 7.4, 3.7, 1.9, 0.93, and 0.02 μM FIS (from left to right). The absorbance at 222 nm was normalized to the maximum peak height for comparison of data at different protein concentrations. The inset in (B) shows a corresponding plot of the change in retention time (•) and width at half height (□) versus FIS concentration.