MAIN-CHAIN DYNAMICS OF CARDIOTOXIN-II FROM TAIWAN COBRA (NAJA-NAJA ATRA) AS STUDIED BY C-13 NMR AT NATURAL-ABUNDANCE - DELINEATION OF THE ROLE OF FUNCTIONALLY IMPORTANT RESIDUES

Cardiotoxin analogue II (TX II) is an all beta-sheet, small molecular mass (6.8 kDa), basic protein possessing a wide array of biological pr operties. Nearly complete assignment of the protonated carbon resonanc es has been achieved by heteronuclear NMR experiments. The study shows that the correlation between the carbon-13 chemical shifts and CTX II structure is good in general, but interesting deviations are also not iced. To characterize the internal dynamics of CTX II, longitudinal, t ransverse relaxation rates and heteronuclear C-13{H-1} NOEs were measu red for alpha-carbons at natural abundance by two-dimensional NMR spec troscopy. Relaxation measurements were obtained in a 14.1 T spectromet er for 50 residues, which are evenly spread along the CTX II polypepti de chain. Except for five alpha-carbons, all data were analysed from a simple two-parameter spectral density function the model free approac h of Lipari and Szabo. The microdynamical parameters (S-2, tau(e), and R-ex) were calculated with an overall rotational correlation time (ta u(m)) for the protein of 4.8 ns. For most residues, the a-carbons exhi bit fast (tau(e) < 30 ps) restricted libration motions (S-2 = 0.79-0.8 9), The present study reveals that the functionally important residues located at the tips of the three loops are flexible, and the flexibil ity of residues in this region could be important in the binding of ca rdiotoxins to their putative ''receptors'' which are postulated to be located on the erythrocyte membrane. In addition, the results obtained in the present study support the earlier predictions on the relative role of the lysine residues in the erythrocyte lytic activity of cardi otoxins.