BIOT 22 |
| 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 small angle X-ray scattering. Overall, equilibrium denaturation data is consitent with a two-state unfolding mechanism with small, but detectable structural changes in the pre-transition region. 1H-15N HSQC cross-peaks for the folded state show only limited chemical shift change as a function of 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 non-specific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of the denaturant binding in inducing the pre-transitional changes is discussed. Experiments on isotopically enriched preparations of IL-1ra show measurable isotopic effects on protein conformational stability in the absence of structural changes. Elucidation of the mechanism of the isotopic effects and their impact on protein folding is currently underway. |
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Biophysical and Biomolecular Symposium: Protein Folding & Characterization
8:00 AM-11:10 AM, Sunday, August 19, 2007 BCEC -- 106, Oral
Division of Biochemical Technology |