On the problem of resonance assignments in solid state NMR of uniformly ¹⁵N,¹³C-labeled proteins

Abstract

Determination of accurate resonance assignments from multidimensional chemical shift correlation spectra is one of the major problems in biomolecular solid state NMR, particularly for relative large proteins with less-than-ideal NMR linewidths. This article investigates the difficulty of resonance assignment, using a computational Monte Carlo/simulated annealing (MCSA) algorithm to search for assignments from artificial three-dimensional spectra that are constructed from the reported isotropic (15)N and (13)C chemical shifts of two proteins whose structures have been determined by solution NMR methods. The results demonstrate how assignment simulations can provide new insights into factors that affect the assignment process, which can then help guide the design of experimental strategies. Specifically, simulations are performed for the catalytic domain of SrtC (147 residues, primarily β-sheet secondary structure) and the N-terminal domain of MLKL (166 residues, primarily α-helical secondary structure). Assuming unambiguous residue-type assignments and four ideal three-dimensional data sets (NCACX, NCOCX, CONCA, and CANCA), uncertainties in chemical shifts must be less than 0.4 ppm for assignments for SrtC to be unique, and less than 0.2 ppm for MLKL. Eliminating CANCA data has no significant effect, but additionally eliminating CONCA data leads to more stringent requirements for chemical shift precision. Introducing moderate ambiguities in residue-type assignments does not have a significant effect.

Keywords: Automated resonance assignments; Monte Carlo algorithm; Solid state NMR.

Figure 1

Flow chart for the Monte Carlo/simulated annealing algorithm for resonance assignment, as contained in the program mcassign2b.

Figure 2

Cartoon representations of the structures of B. anthracis SrtC catalytic domain [44] (a) and MLKL N-terminal domain [45] (b), as determined by solution NMR (PDB 2LN7 and PDB 2MSV, respectively). Chemical shift values for these two proteins (BMRB 18152 and BMRB 25135) were used to generate artificial 3D solid state NMR spectra for assignment simulations. Each protein is viewed from two directions, approximately 90° apart. Signals from segments in grey were taken to be “invisible” in certain simulations, as discussed in the text.

Figure 3

Simulated 2D N-Cα (a) and Cα-Cβ (b) spectra of SrtC and MLKL.