Characterization of the binding of alpha-bungarotoxin to bacterially expressed cholinergic binding sites.
Bacterially expressed cDNA fragments of the alpha-subunit of the nicotinic acetylcholine receptor previously have been shown to bind alpha-bungarotoxin (Gershoni, J. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4318-4321). Here, a novel system has been developed in which totally synthetic alpha-bungarotoxin binding sites are expressed in Escherichia coli transformants. The amino acid sequences, alpha 184-200 and alpha 184-196 of the Torpedo californica alpha-subunit of the nicotinic acetylcholine receptor were expressed as trpE fusion proteins via the expression vector pATH2 and a method for the enrichment of these fusion proteins is described. Quantitative analysis of toxin binding to the recombinant binding sites demonstrates that they bind toxin with affinities of KD = 2.5 X 10(-7) and 4.7 X 10(-6) M, respectively. Furthermore, the pharmacological profile of alpha 184-200 qualitatively reflects that of the intact receptor. These data not only indicate that the area of alpha 184-200 is an essential element of the cholinergic binding site but that residues alpha 197-200 contribute a point of contact between the receptor and alpha-bungarotoxin.
Aronheim
A
A
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Eshel
Y
Y
Mosckovitz
R
R
Gershoni
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014101:Torpedo / Q000378:metabolism; D014170:Transformation, Genetic
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA