Rapid NMR assignments of intrinsically disordered proteins using two-dimensional¹³C-detection based experiments

Abstract

An approach for rapid resonance assignments in proteins based on 2D13C-detected NMR experiments combined with amino acid selective unlabeling.

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions

“…The amino acid mixture was used for initial implementation and evaluation of the sequence, and fully decoupled, very sharp lines of less than 1Hz ( 13 Cα) and 2.5Hz ( 1 Hα) could be obtained (Figure S1). upper and lowercase letters, R: BURBOP refocusing pulse covering the full 13 C-bandwidth, [46][47] I: a BIP 48 or BIBOP [49][50][51] inversion pulse covering the full 13 C and the 15 N amide bandwidth, e: EBURP excitation pulse 52 , r: REBURP refocusing pulse 45,52 . Delay Δ is set to 1/(4 1 J CH ), while Δ 2 is set to 1/(4 1 J CH ) for residues with CH groups in the α-position and 1/(8 1 J CH ) is the optimized value for -CH 2 -groups.…”

“…NMR spectra were recorded at 303K on a 600MHz Bruker Avance III spectrometer equipped with a 5mm 1 H/ 13 C/ 15 N TCI cryogenically cooled probehead, and a Bruker Avance III spectrometer operating at 700.05MHz, equipped with a 5mm Prodigy TCI H&F-C/N-D, zgradient probehead. A commercially available U- 13 C/ 15 N labeled amino acid mixture sample containing variable amounts from each amino acid (Sigma Aldrich) was measured in D 2 O. The composition of the U- 13…”

“…A closer examination of the SHACA-HSQC spectra for both IDPs shows that 1 H and 13 C signal widths (SW) are relatively uniform, being 7-9 Hz and 6-8 Hz, respectively. Knowing that the typical signal dispersion (SD) of an IDP is within 1 ppm ( 1 Hα) and 16 ppm ( 13 Cα, excluding glycines), the corresponding average resolution of SHACA-HSQC is comparable to that of the 13 C-detected CON and it is higher than the average resolution of the 1 H, 15 N-HSQC experiments (see Table 1). 4a).…”

“…In general, decoupling during t 1 is applied without noticeable signal loss and will always lead to an increased signal-to-noise ratio. 13 C-decoupling during t 1 (light gray), 1 H, 13 C, 15 N-decoupling during t 1 (gray), and full decoupling in both dimensions (black) acquired on the free amino acid, p53TAD (peak overlap with a neighboring signal for p53 is indicated by an asterisk), His-Ubi and AS.…”

“…Common Hαdetected experiments suffer from broad signal widths in both 1 H and 13 C dimensions, leading to low resolution and low sensitivity. Surprisingly, it turns out that the signal widths for highly flexible 13 C, 15 N-labeled IDPs is almost exclusively caused by very large multiplets, while underlying natural linewidths are generally narrow. Hence, our aim was to explore this property and to provide a widely applicable, extensively decoupled Hα-detected "pure shift" acquisition scheme with both vastly increased sensitivity and resolution.…”