Engineering a uniquely reactive thiol into a cysteine-rich peptide.
Cysteine mutagenesis for the purpose of chemical labelling was applied to the K+ channel neurotoxin charybdotoxin, a 37-residue peptide with six functionally essential cysteines. An additional 'spinster cysteine' was introduced at a position far away in space from the toxin's known interaction surface where it contacts its K+ channel receptor. Despite the presence of the extra unpaired cysteine residue, the toxin still folds efficiently and may be labelled by fluorescent and radioactive reagents to give a functionally competent toxin.
Shimony
E
E
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, MA.
Sun
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T
Kolmakova-Partensky
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L
Miller
C
C
D000818:Animals; D002478:Cells, Cultured; D018999:Charybdotoxin; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D008958:Models, Molecular; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics; D010084:Oxidation-Reduction; D017354:Point Mutation; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000502:physiology; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents
D009498:Neurotoxins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D012604:Scorpion Venoms; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D018999:Charybdotoxin; C005358:tetramethylrhodamine; D003545:Cysteine