Characterization of the disordered‐to‐α‐helical transition of IA₃ by SDSL‐EPR spectroscopy

Abstract

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA 3 is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA 3 has 68 amino acids, is unstructured in solution, and becomes a-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h (11) /h (0) , the local tumbling volume (V L ), and the percent change of the h (21) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h (11)/ h (0) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA 3 . The other parameters provide sitespecific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of a-helical propensity of the N-terminal segment of IA 3 . Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.

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

“…In the 30% TFE solution, IA 3 becomes helical, and due to the ordered helical backbone restricting the spin-label motion an increase in the spectral correlation time increases from 0.3 ns in the unfolded state to 0.6 ns in the folded state is observed. Corresponding spectral simulations are included in Figure S7, where the slower motion is reflected in a broadening of the spectra, consistent with our previous Xband investigations [37,43]. This spectral broadening impacts overall signal and is reflected in reduced SNR in the 30% TFE samples compared to the 0% TFE samples ( Figure 3A).…”

“…Protein expression, purification and spin-labeling of the E10C IA 3 construct proceeded as described previously [37,43]. Briefly, protein expression was induced by Isopropyl-β-Dthiogalactopyranoside (IPTG); which was optimized by pilot expression and monitoring the intensity of the target protein band intensity in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE).…”

“…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.…”

“…A prime example of X-band EPR studies that fall within this "fast-motion" regime is the characterization of dynamics of intrinsically disordered proteins (IDPs) [37]. IDPs are described as proteins or protein segments that lack a well-defined secondary or tertiary structure [44][45][46][47][48].…”