Effect of D57N mutation on membrane activity and molecular unfolding of cobra cardiotoxin.
Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-strand structure with continuous hydrophobic patch that is capable of interacting with zwitterionic phospholipid bilayer. In addition to the four disulfide bonds that form the rigid core of CTXs, Asp57 near the C-terminus interacts electrostatically with Lys2 near the N-terminus (Chiang et al. 1996. Biochemistry. 35:9177-9186). We indicate herein, using circular dichroism and the time-resolved polarized tryptophan fluorescence measurement, that Asp57 to Asn57 (D57N) mutation perturbs the structure of CTX molecules at neutral pH. The structural stability of the D57N mutant was found to be lower, as evidenced by the reduced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutation also allows a greater degree of molecular unfolding, because the rotational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic interaction between N- and C-termini also contributes to the formation of the functionally important continuous hydrophobic stretch on the distant end of CTX molecules, because both the binding to anilinonaphthalene fluorescent probe and the interaction with phospholipid bilayer were also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a continuous part of the three-fingered beta-strand CTX molecule and indicates how a distant electrostatic interaction might be involved. It is also implicated that electrostatic interaction plays a role in expanding the radius of gyration of the folding/unfolding intermediate of proteins.
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Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan.
Hsu
J H
JH
Sheu
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Chiang
C M
CM
Wu
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Fann
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Tsao
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D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011138:Polytetrafluoroethylene / Q000494:pharmacology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000378:metabolism*; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D004179:Cobra Cardiotoxin Proteins; D005456:Fluorescent Dyes; D013109:Sphingomyelins; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D011138:Polytetrafluoroethylene