Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy.
We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.
Morin
Benjamin
B
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Universités Aix-Marseille I et II, Campus de Luminy, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
Bourhis
Jean-Marie
JM
Belle
Valérie
V
Woudstra
Mireille
M
Carrière
Frédéric
F
Guigliarelli
Bruno
B
Fournel
André
A
Longhi
Sonia
S
D001703:Biophysics / Q000379:methods*; D002627:Chemistry, Physical / Q000379:methods*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D008280:Magnetics; D008968:Molecular Conformation; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels
D010750:Phosphoproteins; D011506:Proteins; D013113:Spin Labels; D009569:Nitric Oxide