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Mutagenesis of the N-terminal cap in the PAS domain of the oxygen transducer, Aer, in Escherichia coli

Taylor, B.L.; Sommer, K.; Johnson, M.S.

FASEB Journal 17(4-5): Abstract 848

2003


ISSN/ISBN: 0892-6638
Accession: 035356313

Aerotaxis is a behavioral response to oxygen in E. coli. The aerotaxis receptor, Aer, has a PAS domain that binds FAD, a membrane-binding domain, a HAMP domain and a signaling domain. PAS domains comprise a sensory protein superfamily. Site-specific mutagenesis of 50 residues in the PAS domain of Aer revealed important residues for FAD binding and signaling. The present study was to investigate the importance of the N-terminal 22 amino-acid Cap of this PAS domain. Residues were chosen for mutagenesis based on a predicted three-dimensional model. These residues were mutagenized to all possible amino acids using doped as well as degenerate primers. Mutations at 9 residues altered the behaviorial phenotype. Mutations Leu14, Thr19 and Leu20 inverted the response to oxygen. That is, cells were repelled by oxygen and attracted to anoxia. Mutations at Pro5, Thr19, Leu20, Met21 and Ser22 abolished aerotactic behavior. Cysteine replacements at 10 residues were able to crosslink in vivo with their cognate residues in a homodimer in response to an oxidant, indicating close proximity between the two PAS domains. The data suggest that the N-terminal Cap can stabilize the structure of the PAS domain and assist signaling between the PAS and HAMP domain. This is the first time that signaling properties have been correlated with specific residues in the N-terminal cap of a PAS domain.

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