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

Study Summary

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

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1ETX 2000-10-11 1.9 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT Q74A
1FIP 1995-02-14 1.9 THE STRUCTURE OF FIS MUTANT PRO61ALA ILLUSTRATES THAT THE KINK WITHIN THE LONG ALPHA-HELIX IS NOT DUE TO THE PRESENCE OF THE PROLINE RESIDUE
1ETO 2000-10-11 1.9 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT R71L
1ETV 2000-10-11 2.0 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT G72A
1ETW 2000-10-11 2.0 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT G72D
1ETY 2000-10-11 2.0 THE CRYSTAL STRUCTURE OF E. COLI WILD-TYPE FIS
1FIA 1993-10-31 2.0 CRYSTAL STRUCTURE OF THE FACTOR FOR INVERSION STIMULATION FIS AT 2.0 ANGSTROMS RESOLUTION
1ETK 2000-10-11 2.1 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT Q68A
3FIS 1993-10-31 2.3 THE MOLECULAR STRUCTURE OF WILD-TYPE AND A MUTANT FIS PROTEIN: RELATIONSHIP BETWEEN MUTATIONAL CHANGES AND RECOMBINATIONAL ENHANCER FUNCTION OR DNA BINDING
4FIS 1993-10-31 2.3 THE MOLECULAR STRUCTURE OF WILD-TYPE AND A MUTANT FIS PROTEIN: RELATIONSHIP BETWEEN MUTATIONAL CHANGES AND RECOMBINATIONAL ENHANCER FUNCTION OR DNA BINDING
5DS9 2016-07-27 2.56 Crystal structure of Fis bound to 27bp DNA F1-8A (AAATTAGTTTGAATTTTGAGCTAATTT)
5DTD 2016-03-09 2.64 Crystal structure of Fis bound to 27bp DNA F1-8C (AAATTCGTTTGAATTTTGAGCGAATTT)
1F36 1997-12-24 2.65 THE CRYSTAL STRUCTURE OF FIS MUTANT K36E REVEALS THAT THE TRANSACTIVATION REGION OF THE FIS PROTEIN CONTAINS EXTENDED MOBILE BETA-HAIRPIN ARMS
5E3L 2016-03-23 2.66 Crystal structure of Fis bound to 27bp DNA F1-8G (AAATTGGTTTGAATTTTGAGCCAATTT)
5E3N 2016-03-09 2.66 Crystal structure of Fis bound to 27bp DNA F31 (AAATTTGTAGGAATTTTCTGCAAATTT)
6P0S 2019-06-19 2.7 Crystal structure of ternary DNA complex 'FX2' containing E. coli Fis and phage lambda Xis
5E3O 2016-03-09 2.78 Crystal structure of Fis bound to 27bp DNA F32 (AAATTTGGAGGAATTTTCTCCAAATTT)
1ETQ 2000-10-11 2.8 THE CRYSTAL STRUCTURE OF E. COLI FIS MUTANT R71Y
3JRH 2010-04-28 2.88 Crystal structure of Fis bound to 27 bp non consensus sequence DNA F21
5E3M 2016-03-09 2.89 Crystal structure of Fis bound to 27bp DNA F35 (AAATTAGTTTGAATCTCGAGCTAATTT)
3JR9 2010-04-28 2.9 Crystal structure of Fis bound to 27 bp optimal binding sequence F2
3IV5 2010-04-28 2.9 Crystal structure of Fis bound to 27 bp optimal binding sequence F1
3JRF 2010-04-28 3.05 Crystal structure of Fis bound to 27 bp DNA F27 containing a C/G at center
3JRC 2010-04-28 3.08 Crystal structure of Fis bound to 27 bp DNA F29 containing 5 G/Cs at center
3JRB 2010-04-28 3.1 Crystal structure of Fis bound to 27 bp DNA F24 containing T-tract at center
3JRD 2010-04-28 3.1 Crystal structure of Fis bound to 27 bp DNA F25 containing T2A3 sequence at center
3JRG 2010-04-28 3.11 Crystal structure of Fis bound to 27 bp non consensus sequence DNA F18
3JRI 2010-04-28 3.11 Crystal structure of Fis bound to 27 bp non consensus sequence DNA F23
3JRA 2010-04-28 3.11 Crystal structure of Fis bound to 27bp non consensus sequence DNA F6
3JRE 2010-04-28 3.17 Crystal structure of Fis bound to 27 bp DNA F26 containing A-tract at center
6P0U 2019-06-19 3.3 Crystal structure of ternary DNA complex ' FX(1-2)-2Xis' containing E. coli Fis and phage lambda Xis
6P0T 2019-06-19 3.6 Crystal structure of ternary DNA complex 'FX(1-2)-1Xis' containing E. coli Fis and phage lambda Xis

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.8 DNA-binding protein Fis P0CW85 FIS_PROVU
100.0 DNA-binding protein Fis O52537 FIS_KLEPN
98.0 DNA-binding protein Fis C6DIJ6 FIS_PECCP
98.0 DNA-binding protein Fis O52540 FIS_PECCA
98.0 DNA-binding protein Fis Q6DAJ8 FIS_PECAS
98.0 DNA-binding protein Fis B4EX21 FIS_PROMH
98.0 DNA-binding protein Fis P0CW84 FIS_PROHU
98.0 DNA-binding protein Fis B1JKF0 FIS_YERPY
98.0 DNA-binding protein Fis Q665E1 FIS_YERPS
98.0 DNA-binding protein Fis A4THB7 FIS_YERPP
98.0 DNA-binding protein Fis Q1CDT5 FIS_YERPN
98.0 DNA-binding protein Fis A9R1Z3 FIS_YERPG
98.0 DNA-binding protein Fis Q8ZAX8 FIS_YERPE
98.0 DNA-binding protein Fis B2K469 FIS_YERPB
98.0 DNA-binding protein Fis Q1C1P4 FIS_YERPA
98.0 DNA-binding protein Fis A7FDQ0 FIS_YERP3
98.0 DNA-binding protein Fis A1JRL7 FIS_YERE8
99.0 DNA-binding protein Fis Q2NWQ0 FIS_SODGM
99.0 DNA-binding protein Fis A4WF77 FIS_ENT38
99.0 DNA-binding protein Fis Q7N015 FIS_PHOLL
100.0 DNA-binding protein Fis Q3YWY8 FIS_SHISS
100.0 DNA-binding protein Fis P0A6R8 FIS_SHIFL
100.0 DNA-binding protein Fis Q0T029 FIS_SHIF8
100.0 DNA-binding protein Fis Q32B77 FIS_SHIDS
100.0 DNA-binding protein Fis Q31W07 FIS_SHIBS
100.0 DNA-binding protein Fis B2U2N7 FIS_SHIB3
100.0 DNA-binding protein Fis A8GK78 FIS_SERP5
100.0 DNA-binding protein Fis P0A6R9 FIS_SERMA
100.0 DNA-binding protein Fis P0A6R6 FIS_SALTY
100.0 DNA-binding protein Fis P0A6R7 FIS_SALTI
100.0 DNA-binding protein Fis B4TX93 FIS_SALSV
100.0 DNA-binding protein Fis B5BGT9 FIS_SALPK
100.0 DNA-binding protein Fis C0PZT1 FIS_SALPC
100.0 DNA-binding protein Fis A9N889 FIS_SALPB
100.0 DNA-binding protein Fis Q5PJW3 FIS_SALPA
100.0 DNA-binding protein Fis B4SUP1 FIS_SALNS
100.0 DNA-binding protein Fis B4TJV9 FIS_SALHS
100.0 DNA-binding protein Fis B5REY3 FIS_SALG2
100.0 DNA-binding protein Fis B5R1C8 FIS_SALEP
100.0 DNA-binding protein Fis B5FIW4 FIS_SALDC
100.0 DNA-binding protein Fis Q57J83 FIS_SALCH
100.0 DNA-binding protein Fis A9MN98 FIS_SALAR
100.0 DNA-binding protein Fis B5F7P5 FIS_SALA4
100.0 DNA-binding protein Fis A6TES8 FIS_KLEP7
100.0 DNA-binding protein Fis B5XND7 FIS_KLEP3
100.0 DNA-binding protein Fis B7LRN6 FIS_ESCF3
100.0 DNA-binding protein Fis B2VL73 FIS_ERWT9
100.0 DNA-binding protein Fis C5BEX0 FIS_EDWI9
100.0 DNA-binding protein Fis Q1R667 FIS_ECOUT
100.0 DNA-binding protein Fis B1LGM6 FIS_ECOSM
100.0 DNA-binding protein Fis B6I1Y1 FIS_ECOSE
100.0 DNA-binding protein Fis B7NDN9 FIS_ECOLU
100.0 DNA-binding protein Fis P0A6R3 FIS_ECOLI
100.0 DNA-binding protein Fis B1IQ31 FIS_ECOLC
100.0 DNA-binding protein Fis P0A6R4 FIS_ECOL6
100.0 DNA-binding protein Fis Q0TCJ5 FIS_ECOL5
100.0 DNA-binding protein Fis A1AGG1 FIS_ECOK1
100.0 DNA-binding protein Fis A8A573 FIS_ECOHS
100.0 DNA-binding protein Fis B1XHN0 FIS_ECODH
100.0 DNA-binding protein Fis C4ZSZ7 FIS_ECOBW
100.0 DNA-binding protein Fis B7M0X3 FIS_ECO8A
100.0 DNA-binding protein Fis B7N0Q5 FIS_ECO81
100.0 DNA-binding protein Fis B7NLI7 FIS_ECO7I
100.0 DNA-binding protein Fis B5YSY6 FIS_ECO5E
100.0 DNA-binding protein Fis P0A6R5 FIS_ECO57
100.0 DNA-binding protein Fis B7LHW8 FIS_ECO55
100.0 DNA-binding protein Fis B7MC31 FIS_ECO45
100.0 DNA-binding protein Fis B7UJZ2 FIS_ECO27
100.0 DNA-binding protein Fis A7ZSF6 FIS_ECO24
100.0 DNA-binding protein Fis A7MJA9 FIS_CROS8
100.0 DNA-binding protein Fis A8AQG0 FIS_CITK8