Properties of FNR proteins substituted at each of the five cysteine residues.
FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally-related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine-rich N-terminal extension, which may form part of an iron-binding, redox-sensing domain in FNR. Site-directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non-reducing SDS-PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster-migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N-terminus. FNR(27) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non-essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A-substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene-derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild-type protein may carry a 78 Da substituent which could not be removed with dithiothreitol or beta-mercaptoethanol.
Green
J
J
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Sharrocks
A D
AD
Green
B
B
Geisow
M
M
Guest
J R
JR
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D007501:Iron; D003545:Cysteine