Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: Evidence for interaction between the two globular domains

The Ca2+ titration of the N-15-labeled mutant V136G calmodulin has been mon itored using H-1-N-15 HSQC NMR spectra. Up to a [Ca2+]/[CaM] ratio of 2, th e Ca2+ ions bind predominantly to sites I and II on the N-domain in contras t with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca2+. Surprisingly, the Ca2+-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-do main in the wild-type protein. The mutated C-domain is observed as a mixtur e of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations depen dent on the [Ca2+]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca2+ binding in the C-domain is mediated by the integrity of the domain structure. Several NH s ignals from residues in the Ca2+-bound N-domain appear as two signals durin g the Ca2+ titration indicating separate species in slow exchange, and it c an be deduced that these result from the presence and absence of interdomai n interactions in the mutant. It is proposed that an unfolded part of the m utated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca2+ affin ity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca2+-binding site, providing details of the involvement of individ ual residues in the calcium-induced folding reactions.