Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule
| Autor: | Ralph Klinke, Arvia E. Morris, James E. Matsuura, Emory H. Braswell, Richard L. Remmele, Wayne R. Gombotz, Randal R. Ketchem |
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| Rok vydání: | 2001 |
| Předmět: |
Models
Molecular Circular dichroism Protein Folding Light Protein Conformation CD40 Ligand Biophysics Trimer CHO Cells Biochemistry Protein Structure Secondary Protein structure Cricetinae Animals Scattering Radiation Denaturation (biochemistry) Molecular Biology Protein secondary structure Coiled coil Chromatography Calorimetry Differential Scanning Fourier Analysis Chemistry Circular Dichroism Temperature Hydrogen-Ion Concentration Molten globule Protein Structure Tertiary Crystallography Spectrometry Fluorescence Protein folding Dimerization Ultracentrifugation |
| Zdroj: | Archives of biochemistry and biophysics. 392(2) |
| ISSN: | 0003-9861 |
| Popis: | The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region. |
| Databáze: | OpenAIRE |
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