Circular dichroism studies of native and chemically modified Ca2+-dependent protein modulator.
The structural features of the native Ca2+-dependent protein modulator and two chemically modified derivatives, namely, nitrotyrosyl modulator and alkylated modulator, were examined by circular dichroism. The binding of Ca2+ to the native molecule was accompanied by an increase in helical content from 40 to 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. Furthermore, nitrotyrosyl modulator interacts with troponin I only in the presence of Ca2+, as detected by circular dichroism (cd). On the other hand, alkylation of five methionine residues on the modulator with benzyl bromide affects protein conformation, as evidenced by a reduced helical content of only 35%. Alkylated modulator retains the ability of the native protein to bind Ca2+ although the affinity of this derivative for Ca2+ is reduced some three orders of magnitude relative to the native protein, with Kd = 3.2 X 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 three conformational states of the molecule.
D000478:Alkylation; D002118:Calcium; D002147:Calmodulin; D002352:Carrier Proteins; D002942:Circular Dichroism; D008956:Models, Chemical; D011487:Protein Conformation / Q000187:drug effects; D014336:Troponin; D014443:Tyrosine / Q000031:analogs & derivatives; D014508:Urea / Q000494:pharmacology
D002147:Calmodulin; D002352:Carrier Proteins; D014336:Troponin; D014443:Tyrosine; D014508:Urea; D002118:Calcium