Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist

JOURNAL OF MOLECULAR BIOLOGY

卷:368

Article

Latypov, Ramil F. ; Harvey, Timothy S. ; Liu, Dingjiang ; Bondarenko, Pavel V. ; Kohno, Tadahiko ; Fachini, Roger A., II ; Rosenfeld, Robert D. ; Ketchem, Randal R. ; Brems, David N. ; Raibekas, Andrei A.

关键词: protein folding;    stability;    denaturant binding;    spectroscopy;    NMR;   

DOI  :  10.1016/j.jmb.2007.02.031

来源: Elsevier

【 摘 要 】

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD,and 1D and 2D (N-1-N-15 heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural, changes within the pretransition region. The majority of the H-1-N-15 HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95 +/- 0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra. is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1ra suggesting similarity of their free energy landscapes. (c) 2007 Elsevier Ltd. All rights reserved.

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