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 ⌬G H 2 O for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 M, 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 ⌬G 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.

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis

“…Since TTR tetramer dissociation, partial monomer denaturation and amyloidogenesis are associated with several familial gain-of-function proteotoxicity diseases, we set out to examine [36][37][38][39][40]. Systematic urea denaturation studies were therefore carried out as a function of the concentration of TTR.…”

“…Two such small molecules are currently being tested as agents to ameliorate FAP in placebo-controlled clinical trials (see http://www.clinicaltrials.gov/). This approach is expected to be efficacious because kinetic stabilization of tetrameric transthyretin incorporating disease-associated subunits by interallelic trans suppression (the incorporation of T119M TTR subunits) ameliorates FAP symptoms (17,18,20,35).Since TTR tetramer dissociation, partial monomer denaturation and amyloidogenesis are associated with several familial gain-of-function proteotoxicity diseases, we set out to examine [36][37][38][39][40]. Systematic urea denaturation studies were therefore carried out as a function of the concentration of TTR.…”

“…Folding and association energetics can be quantified by globally fitting chaotrope denaturation data obtained at several protein concentrations (36)(37)(38)(39)(40). Systematic urea denaturation studies were therefore carried out as a function of the concentration of TTR.…”

Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae: Different mechanisms, similar activation enthalpies

“…When using phosphate buffer and no Mg 2+ (1 mM EDTA present), the unfolding transition was shown to be biphasic with the population of an intermediate species, which became more apparent as protein concentration decreased, as expected for a monomeric intermediate [33][34][35]. Some studies report the ability of the phosphate group to stabilize native enzyme conformations with substrates that include a phosphate group [29].…”

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

“…Under these conditions, the PhnF monomer-dimer equilibrium shifted toward the dimer with increasing total PhnF concentration, and together with a high protein dimer-DNA binding affinity (K F 1 Ϸ K F 2 Ϸ 1 nM), this led to an effective Hill coefficient of n nearly equal to 2. Importantly, both the dimerization and the DNA binding affinity constants were chosen from a typical range of in vivo parameters for bacterial transcription factors (37)(38)(39)(40), suggesting that the observed sigmoidal binding curves can be explained by dimerization of PhnF alone and do not require further, unknown mechanisms.…”