Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.
The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function.
Lynch
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BA
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Koshland
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DE
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D003432:Cross-Linking Reagents; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism
D003432:Cross-Linking Reagents; D004220:Disulfides; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface; C031959:aspartic acid receptor; D001224:Aspartic Acid; D012436:S-Adenosylmethionine; D003545:Cysteine