Structure and Dynamics of Annexin 12 Bound to a Planar Lipid Bilayer

Site directed spin labeling is used to investigate the protein annexin 12 absorbed on a single planar phospholipid bilayer of approximately $2–3{\mathrm{c}\mathrm{m}}^2$. Electron paramagnetic resonance spectra of nitroxide side chain at several topological sites reveal a conserved tertiary fold of the protein in the absorbed state, in agreement with earlier diffraction results. The angular dependent spectra of the two-dimensional microcrystals are shown to provide information on the degree of ordering of spin labels in a $\alpha$-helix and in turn on the orientation of the $\alpha$-helix with respect to the surface.

Figure 1

Reaction of a methanethiosulfonate spin label reagents 1(a) or 1(b) with a protein sulfhydryl to generate the corresponding side chain designated R1(a) ($Y=\mathrm{H}$) or R1(b) ($Y={\mathrm{C}\mathrm{H}}_3$).

Figure 2

Structural model of annexin 12 [15]. The locations of residues 5, 148, 154, and 213 are indicated by spheres on the alpha carbon atoms. The surface represents the headgroup region of the bilayer and was selected to pass through the ${\mathrm{C}\mathrm{a}}^{2+}$ ions in the crystal structure which are the lipid binding sites.

Figure 3

EPR spectra of annexin 12 with R1(a) at the indicated sites. (a) In solution; (b) adsorbed to a single planar bilayer. R1(a) at sites 5, 148, and 154 are angle independent. For 213 spectra are shown for two values of $\theta$, the polar angle between the field and the bilayer normal.

Figure 4

EPR spectra of annexin 12 with R1(b) at 213 and models for the R1 side chain and its motion on helices. (a) In solution. (b)–(e) Adsorbed to a single planar bilayer oriented at the indicated polar angle ($\theta$) with respect to the field. The red traces are fits to the spectra (cf. text). (f) Model of the 213R1(a) side chain on a helix showing the orientation of the $2p$ orbital of the nitrogen and the helical axis, $Z_h$. (g) Definition of the axis and angles used: $Z_L$ is the (instantaneous) axis of the $p$ orbital of the nitroxide, which moves within a cone; $Z_D$ is the symmetry axis of the anisotropic motion (the director axis); $\overrightarrow{n}$ is the bilayer normal; $\overrightarrow{H}$ is the direction of the static magnetic field; $\gamma \angle (Z_D,Z_L)$; $\zeta \angle (Z_D,\overrightarrow{n})$; $\theta \angle (\overrightarrow{n},\overrightarrow{H})$.