Sequence and context dependence of EF-hand loop dynamics.: An 15N relaxation study of a calcium-binding site mutant of calbindin D9k

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using N-15 spin relaxation measurements on the calcium-free state of the calbindin D-9k mutant (A14D+A15 Delta+P20 Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D-9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand-rather than the loop sequence per se-is the major determinant of loop flexibility in this system.