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Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1993 Jan 15; Vol. 268 (2), pp. 909-16. - Publication Year :
- 1993
-
Abstract
- To study the site by which erabutoxin a (Ea) from Laticauda semifasciata binds to the nicotinic acetylcholine receptor, we mutated most residues that are shared with other curaremimetic toxins and studied the structural and biological consequences of introduced mutations. By site-directed mutagenesis, we changed Ser-8 into Gly (EaS8G), Lys-27 into Glu (EaK27E), Trp-29 into Phe (EaW29F) and His (EaW29H), Asp-31 into His (EaD31H), Phe-32 into Leu (EaF32L), Arg-33 into Lys (EaR33K) and Glu (EaR33E), Gly-34 into Ser (EaG34S), Glu-38 into Gln (EaE38Q) and Lys (EaE38K), Gly-49 into Val (EaG49V), and Leu-52 into Ala (EaL52A). All mutants were homogeneous as judged by various analytical procedures. EaE38Q, EaG49V, and EaL52A bound the nicotinic acetylcholine receptor with apparent Kd values close to 10(-10) M, virtually identical to wild Ea. Therefore, Glu-38, Gly-49, and Leu-52 are not important elements in the expression of curaremimetic function in Ea. Mutations of Phe-32 and Gly-34 provoked a 7-fold affinity decrease, suggesting that these residues moderately contribute to function. The 176-fold affinity decrease due to mutation of Ser-8 may reflect some structural change that operates in the polypeptide chain of the mutant, as detected by circular dichroism. Decreases in affinity by a factor of 175, 67, 46, and 318 were seen upon mutations of Lys-27 into Glu, Trp-29 into Phe, Asp-31 into His, and Arg-33 into Glu, with no concomitant change in secondary structure. These residues appear to be important elements of the curaremimetic function of Ea. Thus, a picture of the contribution of conserved residues to the function of a curaremimetic toxin is proposed on the basis of experimental evidence.
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Binding Sites
Binding, Competitive
Chromatography, High Pressure Liquid
Circular Dichroism
Cloning, Molecular methods
Erabutoxins metabolism
Escherichia coli genetics
Kinetics
Molecular Sequence Data
Oligodeoxyribonucleotides
Protein Conformation
Protein Structure, Secondary
Receptors, Cholinergic metabolism
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Erabutoxins chemistry
Erabutoxins genetics
Genetic Engineering methods
Mutagenesis, Site-Directed
Neuromuscular Nondepolarizing Agents chemistry
Neuromuscular Nondepolarizing Agents metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 268
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8419369