Identification of functionally important helical faces in transmembrane segments by scanning mutagenesis.

AUTOR(ES)

Lee, G F

RESUMO

We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of 54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.

Documentos Relacionados

• Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel
• Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis
• Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis.
• Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.
• Amino acids in pneumolysin important for hemolytic activity identified by random mutagenesis.