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-Ju n 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 co nsidering the protein to approximate a prolate ellipsoid. An analysis of the T-1/T-2 ratios for residues in the well-structured section of t he protein showed that a model for the spectral density function in wh ich the protein is considered to reorient anisotropically fitted the d ata significantly better than an isotropic model. Order parameters (S- 2) in the range 0.7-0.9 were observed for most residues, with lower va lues near the C-terminus, consistent with fraying of the two helices c omprising the coiled-coil. Because nearly all of the N-H vectors have small angles to the long axis of the molecule, there was some uncertai nty in the value of the rotational diffusion coefficient D-par, which describes rotation about the long axis. Thus, an alternative method wa s examined for its ability to provide independent estimates of D-par a nd D-perp (the diffusion coefficient describing rotation about axes pe rpendicular 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 dif fusion coefficients proved to be very dependent on the hydrodynamic mo del used to relate D-t to D-par and D-perp, and consequently the value s 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 conform ational exchange process and destabilizes the dimer, no evidence of in creased backbone mobility in this region was detected, suggesting that this process is confined to the Asn side chain.