[5-fluoro-tryptophan-containing N-terminal domain of the alpha-subunit of the Torpedo californica acetylcholine receptor: preparation in E. coli and 19F NMR study].
A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.
Alekseev
T A
TA
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia. t_alex@freemail.ru
Dergousova
N I
NI
Shibanova
E D
ED
Azeeva
E A
EA
Kriukova
E V
EV
Balashova
T A
TA
Dubovskiĭ
P V
PV
Aesen'ev
A S
AS
Tsetlin
V I
VI
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D002846:Chromatography, Affinity; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005109:Extracellular Matrix / Q000378:metabolism; D005461:Fluorine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014101:Torpedo / Q000235:genetics*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D021122:Protein Subunits; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D005461:Fluorine; C026927:5-fluorotryptophan; D012967:Sodium Dodecyl Sulfate; C025154:alpha-cobratoxin; D014364:Tryptophan