Backbone dynamics of the c-Jun leucine zipper: 15N NMR relaxation studies.
The backbone dynamics of the coiled-coil leucine zipper domain of c-Jun have been studied using proton-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation times, together with {1H}15N NOEs, were measured and analyzed by considering the protein to approximate a prolate ellipsoid. An analysis of the T1/T2 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 (S2) 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 Dpar, which describes rotation about the long axis. Thus, an alternative method was examined for its ability to provide independent estimates of Dpar and Dperp (the diffusion coefficient describing rotation about axes perpendicular to the long axis); the transitional diffusion coefficient (Dt) of the protein was measured, and hydrodynamic calculations were used to predict Dpar and Dperp. However, the derived rotational diffusion coefficients proved to be very dependent on the hydrodynamic model used to relate Dt to Dpar and Dperp, and consequently the values obtained from the T1/T2 analysis were used in the order-paramenter 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.
MacKay
J P
JP
Department of Biochemistry, University of Sydney, Australia.
Shaw
G L
GL
King
G F
GF
D000595:Amino Acid Sequence; D016350:Leucine Zippers / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins