Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.
Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) 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 N(TAIL) 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 N(TAIL) 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 N(TAIL) region undergoing alpha-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 alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.
Belle
Valérie
V
Bioénergétique et Ingénierie des Protéines, UPR 9036 CNRS et Université Aix-Marseille I et II, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France.
Rouger
Sabrina
S
Costanzo
Stéphanie
S
Liquière
Elodie
E
Strancar
Janez
J
Guigliarelli
Bruno
B
Fournel
André
A
Longhi
Sonia
S
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D013395:Sucrose; D013696:Temperature; D014270:Trifluoroethanol / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D050505:Mutant Proteins; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D013113:Spin Labels; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus; D013395:Sucrose; D014270:Trifluoroethanol; D003545:Cysteine