NMR Backbone Assignment of Large Proteins by Using 13Cα‐Only Triple‐Resonance Experiments. Issue 28 (8th June 2016)

• Record Type: Journal Article
• Authors: Wei, Qingtao; Chen, Jiajing; Mi, Juan; Zhang, Jiahai; Ruan, Ke; Wu, Jihui
• Is Part Of: Chemistry. Volume 22:Issue 28(2016)
• Page Start: 9556
• Page End: 9564
• Publication Date: 2016-06-08
• Subjects: backbone assignment -- covariance NMR -- isotope labeling -- NMR spectroscopy -- proteins; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.201601871
• Languages: English
• ISSNs: 0947-6539

Abstract

Abstract: Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical‐shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the 13 Cα nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical‐shift displacements, we correctly identify the fidelity of approximately 92 % cross‐peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross‐peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue‐by‐residue study of the backbone structure and dynamics of large proteins. Abstract : A highly sensitive assignment : NMR backbone assignment with a high accuracy of 97.7 % is accomplished by using only HN(CO)CA and HNCA spectra, which show a very high sensitivity even for large proteins. Over 90 % of 13 Cα chemical‐shift degenerated peaks can be discriminated by using peak linear correlation coefficients (LCCs), which are sensitive even to tiny frequency displacements (see picture).