Taking Simultaneous Snapshots of Intrinsically Disordered Proteins in Action

Abstract

Intrinsically disordered proteins (IDPs) as well as intrinsically disordered regions (IDRs) of complex protein machineries have recently been recognized as key players in many cellular functions. NMR represents a unique tool to access atomic resolution structural and dynamic information on highly flexible IDPs/IDRs. Improvements in instrumental sensitivity made heteronuclear direct detection possible for biomolecular NMR applications. The CON experiment has become one of the most useful NMR experiments to get a snapshot of an IDP/IDR in conditions approaching physiological ones. The availability of NMR spectrometers equipped with multiple receivers now enables the acquisition of several experiments simultaneously instead of one after the other. Here, we propose several variants of the CON experiment in which, during the recovery delay, a second two-dimensional experiment is acquired, either based on ¹H detection (CON//HN) or on ¹⁵N detection (CON//btNH, CON//(H)CAN). The possibility to collect simultaneous snapshots of an IDP/IDR through different two-dimensional spectra provides a novel tool to follow chemical reactions, such as the occurrence of posttranslational modifications, as well as to study samples of limited lifetime such as cell lysates or whole cells.

Figure 1

(A) Scheme of the CON//HN experiment. (B) HN and (C) CON 2D spectra acquired through the CON//HN experiment on ¹³C, ¹⁵N-labeled α-synuclein 1 mM at 285 K in 100 mM NaCl, 50 μM EDTA, and 20 mM phosphate buffer (pH 6.5) are shown. (D) A projection of the HN and the (E) CON spectra acquired with the CON//HN experiment (red solid traces) and with the corresponding experiments acquired independently (blue dotted traces) is shown. To see this figure in color, go online.

Figure 2

Comparison of the 2D spectra (HN left; CON right) acquired through the CON//HN experiment on ¹³C, ¹⁵N-labeled α-synuclein at 310 K in different experimental conditions: (A) purified sample in 100 mM NaCl, 50 μM EDTA, and 20 mM phosphate buffer at pH 7.4 is shown; (B) in Escherichia coli cells lysate resuspended in the same buffer as in (A) and (C) in-cell. The traces of a representative signal extracted from the HN (red; left) and the CON (blue; right) spectra are also reported. To see this figure in color, go online.

Figure 3

(A) Scheme of the CON//btNH experiment. The 2D spectra (btNH top; CON bottom) acquired with the CON//btNH experiment on ¹³C,¹⁵N-labeled α-synuclein 1 mM at (B) 285 K and (C) 315 K at pH 6.5 in the same buffer reported in Fig. 1 are shown. (D) and (E) report the comparison of the S/N of the projection of the spectra acquired with the CON//btNH experiment (red solid traces) with the analogous spectra acquired independently (blue dotted traces). To see this figure in color, go online.

Figure 4

(A) Scheme of the CON//(H)CAN experiment. (B) (H)CAN and (C) CON spectra recorded on ¹³C, ¹⁵N-labeled α-synuclein at 310 K (pH 7.4) are shown. (D) and (E) report the comparison of the S/N of the projection of the spectra acquired with the CON//(H)CAN experiment (red solid traces) with the analogous spectra acquired independently (blue dotted traces). To see this figure in color, go online.

Figure 5

The 2D spectra acquired with the CON//HN experiment to monitor phosphorylation of ¹³C, ¹⁵N-labeled α-synuclein 200 μM with Fyn tyrosine kinase as a function of time (t = 0 h, red spectra; t = 50 h blue spectra). The HN spectra are reported on the left and the CON on the right. The highlighted region of the CON spectra is enlarged to show, as an example, a subset of α-synuclein signals that are influenced by phosphorylation. The reaction was performed at 303 K in 20 mM phosphate buffer, 100 mM NaCl, 50 μM EDTA, 2 mM dithiothreitol, 6 mM MgCl₂, and 3 mM ATP (pH 7.0). To see this figure in color, go online.