Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy
| Autor: | André Fournel, Sonia Longhi, Stéphanie Costanzo, Janez Strancar, Sabrina Rouger, Elodie Liquière, Valérie Belle, Bruno Guigliarelli |
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| Rok vydání: | 2008 |
| Předmět: |
Protein Folding
Sucrose Crystallography X-Ray Biochemistry Protein Structure Secondary law.invention Viral Proteins Structural Biology law Cysteine Electron paramagnetic resonance Molecular Biology Protein secondary structure Measles Virus Nucleoprotein Chemistry C-terminus Circular Dichroism Electron Spin Resonance Spectroscopy Temperature Site-directed spin labeling Trifluoroethanol Nucleocapsid Proteins Nucleoprotein Protein Structure Tertiary Folding (chemistry) Crystallography Nucleoproteins Amino Acid Substitution Phosphoprotein Electrophoresis Polyacrylamide Gel Mutant Proteins Spin Labels |
| Zdroj: | Proteins. 73(4) |
| ISSN: | 1097-0134 |
| Popis: | Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (NTAIL) that undergoes induced folding. In addition to four spin-labeled NTAIL variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of NTAIL in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of NTAIL were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488–502 and the 505–522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488–502, the NTAIL region undergoing α-helical folding. The mobility of the 488–502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505–522 region upon binding to XD is due to the α-helical transition occurring within the 488–502 region and not to a direct interaction with XD. Proteins 2008. © 2008 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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