The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis.
The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain. The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues. Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not. The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues. However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate. These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence. This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics.
Jeffery
C J
CJ
Department of Molecular and Cell Biology, 329 Stanley Hall, University of California, Berkeley, CA 94720, USA.
Koshland
D E
DE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D017459:Receptors, Amino Acid / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism
D009841:Oligonucleotides; D017459:Receptors, Amino Acid; D011994:Recombinant Proteins; C031959:aspartic acid receptor; D014867:Water; D001224:Aspartic Acid; D011972:Receptor, Insulin