Abstract
Cobra cardiotoxins (CTXs) are able to adopt a three-fingered β-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, Asp 57 near the C-terminus interacts electrostatically with Lys 2 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 Asp 57 to Asn 57 (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 β-sheet to α-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 β-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.