Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd.
A novel experiment has been used to show proximity relationships between sites on the surface of the cytochrome bd quinol oxidase of Escherichia coli. The artificial protease iron (S)-1-[p-(bromoacetamido)benzyl]-EDTA (Fe--BABE) was conjugated to selected reactive cysteines placed in subunit I or subunit II, with the aim of identifying amino acid residues within approximately 12 A of each site of attachment. The protease was activated with H2O2 and ascorbate for a few seconds, and hydrolysis products were isolated and analyzed by N-terminal sequencing. Among other results, we found that residue 39 of subunit II is near residue 255 of subunit I in the putative quinone-binding domain (Q loop) of the oxidase. Since this technique is insensitive to the nature of the amino acid side chains, it should prove generally valuable in revealing spatial relationships both within and between subunits in complex proteins where high-resolution structural information is not available.
Ghaim
J B
JB
School of Chemical Sciences, University of Illinois, Urbana 61801,USA.
Greiner
D P
DP
Meares
C F
CF
Gennis
R B
RB
D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D045222:Electron Transport Chain Complex Proteins; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding
D016227:Benzoquinones; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009942:Organometallic Compounds; C004532:quinone; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D010450:Endopeptidases