Structural characterization of intrinsically disordered proteins by NMR spectroscopy

Abstract

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs.

Figure 1

TROSY-¹⁵N-HSQC (left) and CON (right) spectra of the fully disordered protein ERD14. The comparison of both spectra clearly shows an improvement on the chemical shift dispersion by going from ¹H- to ¹³C-detected experiments: While in the ¹⁵N-HSQC all the ¹H frequencies are clustered within an area of 1 ppm (8.6–7.6 ppm region), ¹³C’ resonances are still distributed over the range of 7 ppm (176–169 ppm). Acquisition of the spectra was done at 600 MHz Bruker spectrometer at 15 °C by using a CryoProbe TCI. Estimated protein concentration 50 μM (10 mM MES, pH 6.5).

Figure 2

Paramagnetic probes for long-range contacts. If long-range contacts are present, residues sequentially distant from the tag will experience the PRE effect more frequently than would be expected from ideal random coil behavior. Different conformers will experience different PREs up to a range of 25Å as indicated by red spheres.

Figure 3

Correlations observed in CON based experiments. Schematic representation of the detected correlations by using the following ¹³C-direct detection NMR experiment type: 3D COCON experiment (purple), 3D CBCACON (orange) and 2D CON (green).