Backbone dynamics of the c-Jun leucine zipper: N-15 NMR relaxation studies

The backbone dynamics of the coiled-coil leucine zipper domain of c-Jun have been studied using proton-detected two-dimensional H-1-N-15 NMR spectroscopy. Longitudinal (T-1) and transverse (T-2) N-15 relaxation times, together with {H-1}N-15 NOEs, were measured and analyzed by considering the protein to approximate a prolate ellipsoid. An analysis of the T-1/T-2 ratios for residues in the well-structured section of the protein showed that a model for the spectral density function in which the protein is considered to reorient anisotropically fitted the data significantly better than an isotropic model. Order parameters (S-2) in the range 0.7-0.9 were observed for most residues, with lower values near the C-terminus, consistent with fraying of the two helices comprising the coiled-coil. Because nearly all of the N-H vectors have small angles to the long axis of the molecule, there was some uncertainty in the value of the rotational diffusion coefficient D-par, which describes rotation about the long axis. Thus, an alternative method was examined for its ability to provide independent estimates of D-par and D-perp (the diffusion coefficient describing rotation about axes perpendicular to the long axis); the translational diffusion coefficient (D-t) of the protein was measured, and hydrodynamic calculations were used to predict D-par and D-perp. However, the derived rotational diffusion coefficients proved to be very dependent on the hydrodynamic model used to relate D-t to D-par and D-perp, and consequently the values obtained from the T-1/T-2 analysis were used in the order-parameter analysis. Although it has previously been reported that the side chain of a polar residue at the dimer interface, Asn22, undergoes a conformational exchange process and destabilizes the dimer, no evidence of increased backbone mobility in this region was detected, suggesting that this process is confined to the Asn side chain