Backbone Dynamics of the c-Jun Leucine Zipper: 15N NMR Relaxation Studies

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• 作者：Joel P. Mackay ; Graeme L. Shaw ; and Glenn F. King
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：April 16, 1996
• 年：1996
• 卷：35
• 期：15
• 页码：4867 - 4877
• 全文大小：765K
• 年卷期：v.35,no.15(April 16, 1996)
• ISSN：1520-4995

文摘

The backbone dynamics of the coiled-coil leucinezipper domain of c-Jun have been studiedusing proton-detected two-dimensional ¹H-¹⁵NNMR spectroscopy. Longitudinal (T₁) andtransverse(T₂) ¹⁵N relaxation times, togetherwith {¹H}¹⁵N NOEs, were measured andanalyzed by considering theprotein to approximate a prolate ellipsoid. An analysis of theT₁/T₂ ratios for residuesin the well-structuredsection of the protein showed that a model for the spectral densityfunction in which the protein is consideredto reorient anisotropically fitted the data significantly better thanan isotropic model. Order parameters(S²) in the range 0.7-0.9 were observed formost residues, with lower values near the C-terminus,consistentwith fraying of the two helices comprising the coiled-coil.Because nearly all of the N-H vectors havesmall angles to the long axis of the molecule, there was someuncertainty in the value of the rotationaldiffusion coefficient D_par, which describesrotation about the long axis. Thus, an alternative methodwasexamined for its ability to provide independent estimates ofD_par and D_perp (thediffusion coefficientdescribing rotation about axes perpendicular to the long axis); thetranslational diffusion coefficient(D_t)of the protein was measured, and hydrodynamic calculations were used topredict D_par and D_perp.However,the derived rotational diffusion coefficients proved to be verydependent on the hydrodynamic modelused to relate D_t to D_parand D_perp, and consequently the values obtainedfrom the T₁/T₂ analysiswereused in the order-parameter analysis. Although it has previouslybeen reported that the side chain of apolar residue at the dimer interface, Asn22, undergoes a conformationalexchange process and destabilizesthe dimer, no evidence of increased backbone mobility in this regionwas detected, suggesting that thisprocess is confined to the Asn side chain.