Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence.

Addition of calmodulin to caldesmon causes a concentration-dependent shortwave shift and an increase of fluorescence intensity of caldesmon tryptophan residues. The existence of a protein complex is confirmed by the increase of the caldesmon sedimentation coefficient s0(20,w) from 3.0 S to 4.5 S in the presence of calmodulin. These findings allow application of the method of protein intrinsic tryptophan fluorescence for quantitative study of unmodified caldesmon and calmodulin in solution. The affinity of the caldesmon-calmodulin interaction (Kass = 1.8 x 10(6) M-1, in 0.1 M-KCl at 25 degrees C) and Ca2+ requirement (half-maximum binding at 0.8 microM-Ca2+) determined by means of the fluorescence technique are in agreement with previously reported values, thus confirming the validity of the method. The same approach was further used to provide information about the nature of interactions stabilizing the caldesmon-calmodulin complex. Association of the proteins and dissociation of the complex were studied in different physicochemical conditions, including variation of pH, temperature and ionic strength and the addition of quenchers, denaturants and anticalmodulin drugs. The results obtained suggest that caldesmon tryptophan residues, together with charged groups, are involved in calmodulin binding. Hydrophobic, electrostatic and hydrogen interactions contribute to the stability of the protein complex, making it insensitive to variations of physicochemical conditions within physiological limits.