Complete Assignment of Heteronuclear Protein Resonances by Protonless NMR Spectroscopy

Abstract

The complete assignment of the resonances of a protein is key to the determination of its solution structure by NMR spectroscopy and for the study of protein-protein and protein-ligand interactions. The proton-based assignment strategy usually starts with the correlation of individual resonances of each amino acid residue through scalar connectivities followed by linking them one after the other. [1,2] Although many different triple-resonance NMR spectroscopy experiments have been designed for full assignments, [2] spectral overlap can still lead to ambiguities. This poses a significant limiting factor in the cases of large and/or paramagnetic biomolecules. [3] After the pioneering report of 13 C NMR spin-system assignments of 13 C-enriched Anabaena 7120 ferredoxin by Markley and co-workers, [4] heteronuclear NMR spectroscopy experiments were progressively abandoned in favor of 1 Hdetection experiments. However, as was recently pointed out, heteronuclear NMR spectroscopy decreases the effect of detrimental transverse relaxation, which is typical of large or paramagnetic proteins. [5][6][7][8][9][10][11][12][13][14][15][16][17] For this reason, several heteronuclear NMR spectroscopy experiments for backbone assignment have been proposed for fully 13 C-and 15 N-enriched proteins. [13,14,17] Furthermore, backbone sequence-specific assignment by the recently-designed CANCO experiment has also been reported. [18] We present herein an extension of the set of exclusively heteronuclear experiments to protein side chain resonances for the complete heteronuclear assignment of a protein. With a novel CBCACO experiment the carbonyl carbon (CO) is linked to the C b and to the C a nuclei; the connection to the rest of the amino acid side chain is achieved through a 13 C-13 C TOCSY experiment with C a detection. In these experiments, we have successfully implemented spin-state selection methods for the removal of signal splitting in the acquisition dimension which is caused by multiple 13 C-13 C scalar couplings. This makes 13 C detection an amenable tool for high-resolution NMR spectroscopy. The proposed assignment strategy is summarized in Figure 1. A Figure 1. Illustration of the assignment procedure for 13 C NMR spectroscopy experiments. The assignment starts with analysis of the CACO experiment, which provides the correlation between the carbonyl carbon (CO) and the C a nuclei of each amino acid. The spin-system assignment is extended to the C b nuclei with the CBCACO experiment, and the process is completed with the TOCSY experiment, which provides correlation between the C a and the other carbon nuclei of the amino acid side chain. The amino acid spin systems are finally assigned in a sequence-specific manner with the aid of a CANCO experiment, [18] which provides the correlation of each CO to the two neighboring C a nuclei.

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and Upon Multisite Phosphorylation

“…For assignment, approximately 0.7 mM 15 N, 13 C Ash1 sample was used to acquire standard triple-resonance backbone assignment experiments and carbon-detect triple resonance experiments. Standard assignment experiments were based on sensitivity enhanced 1 H- 15 65 Additionally, CCCON-IPAP, a 3D 13 C TOCSY, was used to distinguish trans versus cis proline (32 scans, 1024 ( 13 C) × 48 ( 15 N) × 160 ( 13 C) complex data points, with 18 ppm, 36 ppm and 72 ppm as 13 C, 15 N and 13 C (TOCSY) sweep widths). 66 15 N NMR relaxation experiments acquired on a Bruker Avance 800 MHz spectrometer at 278 K using standard pulse programs (16 scans, 2048 ( 1 H) × 150 ( 15 N) complex data points).…”

Molecular Basis of the Mixed Lineage Leukemia-Menin Interaction

“…2A). Therefore, we chose to employ 13 C-detected experiments (20) to map the residues of MLL 46 required for binding to menin. We obtained nearly complete assignment of MLL 46 based on combination of two-dimensional CACO, CANCO, CBCACO, and NCO experiments ( Fig.…”

“…The 13 C-detected experiments are particularly well suited for this purpose due to high chemical shift dispersion and narrow line widths of peaks for highly flexible unfolded proteins (20,27). Contrary to standard HN-detected triple resonance experiments, the 13 C-detected experiments are independent of H-D exchange of amide protons (see supplementary Fig.…”

Singlet states open the way to longer time‐scales in the measurement of diffusion by NMR spectroscopy

“…This approach is particularly effective, as the low gyromagnetic ratios of heteronuclei scale down all dipolar interactions, including those with electronic spins. The use of low-gamma nuclei to increase the lifetimes of spin-order has been predicted to be effective in both paramagnetic and large molecules, and can be introduced in experiments with direct excitation and detection of heteronuclei (13,14).…”