Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins.

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca(2+) binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With (D63N)S100B as an exception ((D63N)K(D)=50±9 μM), Ca(2+) binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 ((D61N)K(D)=412±67 μM, (D65N)K(D)=968±171 μM, and (E72A)K(D)=471±133 μM), when compared to wild-type protein ((WT)K(D)=56±9 μM). For the TRTK-12 complexes, the Ca(2+)-binding affinity to wild type ((WT+TRTK)K(D)=12±10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target ((D61N+TRTK)K(D)=29±1.