Circular dichroism studies of native and chemically modified Ca2+-dependent protein modulator

Abstract

The structural features of the native [bovine brain] Ca2+-dependent protein modulator and 2 chemically modified derivatives, i.e., nitrotyrosyl modulator and alkylated modulator, were examined by circular dichroism (CD). The binding of Ca2+ to the native molecule was accompanied by an increase in helical content from 40-49%, with little effect on the local environments of aromatic residues in the modulator. The Mg2+ and Mn2+ do not elicit the conformational change induced by the binding of Ca2+, which also stabilizes the modulator against urea denaturation. The overall secondary structure of nitrotyrosyl modulator is indistinguishable from that of the native protein and undergoes a similar conformational change upon binding Ca2+. These observations are in agreement with the fact that nitration has no effect on modulator functions. Nitrotyrosyl modulator interacts with troponin I only in the presence of Ca2+, as detected by (CD). Alkylation of 5 methionine residues on the modulator with benzyl bromide affects protein conformation as evidenced by a reduced helical content of only 34%. Alkylated modulator retains the ability of the native protein to bind Ca2+ although the affinity of this derivative for Ca2+ is reduced some 3 orders of magnitude relative to the native protein, with Kd = 3.2 .times. 10-4 M. The results with the alkylated modulator, in conjunction with previous CD studies on N-chlorosuccinimide oxidized modulator are utilized to advance a model for the Ca2+ activation of modulator protein based on 3 conformational states of the molecule.