ABSTRACT: 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 ?M), 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 ?M, (D65N)K(D)=968±171 ?M, and (E72A)K(D)=471±133 ?M), when compared to wild-type protein ((WT)K(D)=56±9 ?M). For the TRTK-12 complexes, the Ca(2+)-binding affinity to wild type ((WT+TRTK)K(D)=12±10 ?M) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target ((D61N+TRTK)K(D)=29±1.2 ?M, (D63N+TRTK)K(D)=10±2.2 ?M, (D65N+TRTK)K(D)=73±4.4 ?M, and (E72A+TRTK)K(D)=18±3.7 ?M). In addition, R(ex), as measured using relaxation dispersion for side-chain (15)N resonances of Asn63 ((D63N)S100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, (D61N)S100B, (D63N)S100B, and (D65N)S100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca(2+)-bound (2.0Å) and TRTK-bound (1.2Å) (D63N)S100B showed no change in Ca(2+) coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca(2+)-binding affinity in solution. Therefore, a model for how S100B-TRTK-12 complex formation increases Ca(2+) binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.