EFFECT OF D57N MUTATION ON MEMBRANE-ACTIVITY AND MOLECULAR UNFOLDING OF COBRA CARDIOTOXIN

Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-stra nd structure with continuous hydrophobic patch that is capable of inte racting with zwitterionic phospholipid bilayer. In addition to the fou r 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 fl uorescence measurement, that Asp(57) to Asn(57) (D57N) mutation pertur bs the structure of CTX molecules at neutral pH. The structural stabil ity of the D57N mutant was found to be lower, as evidenced by the redu ced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutati on also allows a greater degree of molecular unfolding, because the ro tational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic in teraction between N- and C-termini also contributes to the formation o f the functionally important continuous hydrophobic stretch on the dis tant end of CTX molecules, because both the binding to anilinonaphthal ene fluorescent probe and the interaction with phospholipid bilayer we re also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a con tinuous part of the three-fingered beta-strand CTX molecule and indica tes how a distant electrostatic interaction might be involved. It is a lso implicated that electrostatic interaction plays a role in expandin g the radius of gyration of the folding/unfolding intermediate of prot eins.