Role of amino and carboxyl groups of cobrotoxin in the conformational stability and the interaction with acetylcholine receptor.
To study the functional involvements of the common interaction of the Leu-1 alpha-amino group and Asp-58 in cobrotoxin, the lysine epsilon-amino groups of cobrotoxin were initially guanidinated with o-methylisourea. The alpha-amino group of Leu-1 was them modified with TNBS after the guanidination of cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the beta-sheet secondary structure of cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 alpha-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of cobrotoxin, and the free carboxyl groups in cobrotoxin are conformation-essential.
Kuo
K W
KW
Department of Biochemistry, Kaohsiung Medical College, Taiwan, Republic of China.
Chang
L S
LS
Chang
C C
CC
D000588:Amines / Q000378:metabolism; D000818:Animals; D000937:Antigen-Antibody Reactions; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002255:Carbonic Acid / Q000737:chemistry; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D007457:Iodine Radioisotopes; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011863:Radioimmunoassay; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000378:metabolism*; D014101:Torpedo
D000588:Amines; D003039:Cobra Neurotoxin Proteins; D007457:Iodine Radioisotopes; D011950:Receptors, Cholinergic; D002255:Carbonic Acid; C035591:glycine methyl ester; D001216:Asparagine; D005998:Glycine