Backbone Dynamics of the Calcium-Signaling Protein apo-S100B as Determined by 15N NMR Relaxation

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Abstract
Backbone dynamics of homodimeric apo-S100B were studied by 15N nuclear magnetic resonance relaxation at 9.4 and 14.1 T. Longitudinal relaxation (T1), transverse relaxation (T2), and the 15N-{1H} NOE were measured for 80 of 91 backbone amide groups. Internal motional parameters were determined from the relaxation data using the model-free formalism while accounting for diffusion anisotropy. Rotational diffusion of the symmetric homodimer has moderate but statistically significant prolate axial anisotropy (D∥/D⊥ = 1.15 ± 0.02), a global correlation time of τm = 7.80 ± 0.03 ns, and a unique axis in the plane normal to the molecular symmetry axis. Of 29 residues at the dimer interface (helices 1 and 4), only one has measurable internal motion (Q71), and the order parameters of the remaining 28 were the highest in the protein (S2 = 0.80 to 0.91). Order parameters in the typical EF hand calcium-binding loop (S2 = 0.73 to 0.87) were slightly lower than in the pseudo-EF hand (S2 = 0.75 to 0.89), and effective internal correlation times, τe, distinct from global tumbling, were detected in the calcium-binding loops. Helix 3, which undergoes a large, calcium-induced conformational change necessary for target-protein binding, does not show evidence of interchanging between the apo and Ca2+-bound orientations in the absence of calcium but has rapid motion in several residues throughout the helix (S2 = 0.78 to 0.88; 10 ≤ τe ≤ 30 ps). The lowest order parameters were found in the C-terminal tail (S2 = 0.62 to 0.83). Large values for chemical exchange also occur in this loop and in regions nearby in space to the highly mobile C-terminal loop, consistent with exchange broadening effects observed.

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