Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

Abstract

We introduce a Python-based program that utilizes the large database of (13)C and (15)N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D (13)C-(13)C, (15)N-(13)C, or 3D (15)N-(13)C-(13)C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D (13)C-(13)C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40-60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO-Cα-Cβ or N-Cα-Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

Fig. 1

Selected regions of several 2D ¹³C-¹³C chemical shift correlation maps. a Ala Cα-Cβ. b Gly CO–Cα. c Phe Cα-Cβ. d Arg Cα-CO. The chemical shifts are color-coded in red for helix, green for sheet and blue for coil in this paper. The ranges of the linear contour levels are indicated for each panel. Although there are regions where secondary structures can be discerned unambiguously, the coil chemical shift range overlaps significantly with the helix and sheet chemical shift regions. Dashed boxes in a indicate second maxima in the helix and coil chemical shift distributions of Ala

Fig. 2

2D ¹³C-¹³C correlation maps of Ile and Val. Gray bars denote the methyl Cγ1/2 chemical shifts that can be uniquely assigned to α-helical conformation. The values are greater than 30 ppm for Ile and greater than 23.5 ppm for Val

Fig. 3

2D ¹³C-¹³C correlation maps of Leu and Pro. Note that the Pro chemical shifts are dominated by the coil conformation

Fig. 4

2D ¹³C-¹³C correlation maps of Lys and Arg. Lys exhibits a characteristic pattern of three nearly equidistant Cα-sidechain correlation strips that are orthogonal to the ridge formed by the Cε correlation peaks. Note the bimodal distribution of β-sheet Arg Cα-Cβ chemical shifts

Fig. 5

2D ¹³C-¹³C correlation maps of sidechain-carbonyl-containing Glu and Gln. Despite the similarity of their chemical shifts, the 36–40 ppm region is uniquely Glu Cβ (gray bars). Chemical shifts above 180 ppm generally result from Cδ

Fig. 6

¹³C chemical shift regions that can be uniquely assigned to a single residue type. PLUQ was run for 1 × 1 ppm bins within 5–75 ppm using the “all” options, which give amino acid types irrespective of the secondary structure. A bin was filled if a single residue type was found or if the most probable residue type is more than 10 times more likely than the second most likely amino acid. Only bins with more than 10 data points were considered, to eliminate outliers in the database

Fig. 7

Representative PLUQ queries. (a) Cβ and Cα chemical shifts of a β-sheet Leu with a chemical shift search range (the “-r 1.0” option) of ± 1.0 ppm. (b) CO, Cα and Cβ chemical shifts of a beta-sheet Ala. (c) Cα, Cβ and Cδ chemical shifts of Arg, where all secondary structures were grouped together using the “-c all” option and only the top three most probable amino acid types were outputted using the “–m 3” option. Note that Arg Cα - Cδ assignment was not outputted because the default bond number was 2. It can be changed to 3 using the option “-b 3”