Assessing Induced Folding of an Intrinsically Disordered Protein by Site-Directed Spin-Labeling Electron Paramagnetic Resonance Spectroscopy

Abstract

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.

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

“…4B). Such lineshapes are characteristic of dynamically disordered states (18,60,61), suggesting that the loss of helicity detected by CD under pressure may be attributed in part to regions involving helices E and F. This is in contrast to apoMb at pH 4.1, 2 kbar (see below), where sharp isotropic resonance lines are observed for residues 42R1 and 54R1 as well, 2 and 4, Fig. 4A).…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…For instance, CW spectra at W-and D-bands provide an enhanced capability to detect sub-nanosecond correlation times [43]. This is particularly suitable for studying IDPs, in which the time scale normally falls into the 0.1 to 2 ns region [37,38,[44][45][46][47][48][69][70][71][72].…”

“…Examples of nitroxide spin-labeled molecules falling into a fast motion regime (i.e. correlation times < 2 ns) include small peptides/proteins, small nucleic acids, unstructured proteins/protein segments, and backbone/nucleobase labeling of RNAs [37][38][39][40][41][42]. For these biological systems, it is oftentimes challenging to extract dynamic parameters from the subtle differences of the narrow line shapes of CW EPR spectra at X-band.…”

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus

“…By analogy with the closely related Sendai virus (13,14), it is thought to be tetrameric, contrary to the dimeric P proteins of rabies and vesicular stomatitis viruses (15,16). The P oligomers simultaneously bind to L (via the P multimerization domain) and to the exposed C-terminal domain of N (N TAIL ; amino acids 400 -525) via the C-terminal X domain of P (XD; amino acids 459 -507) (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Progressive movement of the polymerase along its template thus is thought to require cycles of XD-N TAIL binding and release (see Ref.…”

“…N TAIL is an intrinsically disordered domain (12,18) that undergoes ␣-helical folding upon binding to XD (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Structure prediction and available experimental observations indicate that the disordered nature of the C-terminal region of N is a conserved feature within members of the Paramyxovirinae subfamily (29 -32).…”