Structure and Dynamics of a Helical Hairpin and Loop Region in Annexin 12:  A Site-Directed Spin Labeling Study

A 30-residue nitroxide scan encompassing a helical hairpin and an extended loop in soluble annexin 12 (helices D and E in repeat 2; residues 134−163) has been analyzed in terms of nitroxide side chain mobility and accessibility to collision with paramagnetic reagents (Π). Values of Π for both O2 and a Ni(II) metal complex (NiEDDA) are remarkably well correlated with the fractional solvent accessibility of the native side chains at the corresponding positions computed from the known crystal structure. This result demonstrates the utility of Π as an experimental measure of side chain accessibility in solution, as well as the lack of structural perturbation due to the presence of the nitroxide side chain. The pattern of side chain mobility is also in excellent agreement with predictions from the crystal structure. The results presented here extend the correlations between mobility and structure described in earlier work on other helical proteins, and suggest their generality. The periodic dependence of Π and mobility along the sequence of annexin 12 reveals the helical segments and their orientation in the fold, as expected for a nonperturbing nitroxide side chain. However, these data do not distinguish the helix−loop−helix motif from a continuous helix, because immobilized side chains in the short loop sequence maintain the periodicity. As shown here, the ratio of Π values for O2 and NiEDDA clearly delineates the loop region, due to size exclusion effects between the two reagents. A new feature evident in a nitroxide scan through multiple secondary elements is a modulation of the basic Π and mobility patterns along the sequence, apparently due to differences in helix packing and backbone motion. Thus, in the short helix D, residues are consistently more mobile and accessible throughout the sequence compared to the residues in the longer, less-solvated and more ordered helix E.