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. Study holds ProTherm entries: 15253, 15254, 15255, 15256, 15257, 15258, 15259, 15260, 15261, 15262, 15263 Extra Details: far UV-CD at 222 nm Factor for inversion stimulation, FIS; equilibrium denaturation; dimer stability; transcription regulation; dissociation constant; DNA binding

Submission Details

ID: WoEDBCwX

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Hobart SA;Ilin S;Moriarty DF;Osuna R;Colón W,Protein Sci. (2002) Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function. PMID:12070319
Additional Information

Number of data points 33
Proteins DNA-binding protein Fis ; DNA-binding protein Fis
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm buffers:MOPS: 20 mM, prot_conc:107.0 micro M ; Experimental Assay: m buffers:MOPS: 20 mM, prot_conc:107.0 micro M ; Experimental Assay: dG_H2O buffers:MOPS: 20 mM, prot_conc:107.0 micro M ; Experimental Assay: Cm buffers:MOPS: 20 mM, prot_conc:35.6 micro M ; Experimental Assay: m buffers:MOPS: 20 mM, prot_conc:35.6 micro M ; Experimental Assay: dG_H2O buffers:MOPS: 20 mM, prot_conc:35.6 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:107.0 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:35.6 micro M ; Experimental Assay: Cm buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: m buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: dG_H2O buffers:Potassium phosphate: 10 mM, prot_conc:8.9 micro M ; Experimental Assay: Cm prot_conc:1.8 micro M, buffers:Potassium phosphate: 10 mM ; Experimental Assay: m prot_conc:1.8 micro M, buffers:Potassium phosphate: 10 mM ; Experimental Assay: dG_H2O prot_conc:1.8 micro M, buffers:Potassium phosphate: 10 mM
Libraries Mutations for sequence MFEQRVNSDVLTVSTVNSQDQVTQKPLRDSVKQALKNYFAQLNGQDVNDLYELVLAEVEQPLLDMVMQYTRGNQTRAALMMGINRGTLRKKLKKYGMN
Sequence Assay Result Units