Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state.
Fur (ferric uptake regulator) protein is a DNA-binding protein which regulates iron-responsive genes. Recombinant Fur from the nitrogen-fixing cyanobacterium Anabaena PCC 7119 has been purified and characterized, and polyclonal antibodies obtained. The experimental data show that Fur from Anabaena dimerizes in solution with the involvement of disulphide bridges. Cross-linking experiments and MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) MS also show several oligomerization states of Fur, and the equilibrium of these forms depends on protein concentration and ionic strength. In intact recombinant Fur, four cysteine residues out of five were inert towards DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)], and their modification required sodium borohydride. Metal analysis and electrospray ionization MS revealed that neither zinc nor other metals are present in this Fur protein. Purified recombinant Fur bound to its own promoter in gel-shift assays. Fur was shown to be a constitutive protein in Anabaena cells, with no significant difference in its expression in cells grown under iron-sufficient compared with iron-deficient conditions.
Hernández
José A
JA
Departamento de Bioqui;mica y Biologi;a Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009-Zaragoza, Spain.
Bes
M Teresa
MT
Fillat
María F
MF
Neira
José L
JL
Peleato
M Luisa
ML
D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007477:Ions; D007501:Iron / Q000378:metabolism; D007525:Isoelectric Focusing; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D007477:Ions; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013438:Sulfhydryl Compounds; C068477:ferric uptake regulating proteins, bacterial; D004247:DNA; D007501:Iron; D003545:Cysteine