Factors affecting the use of 13Cα chemical shifts to determine, refine, and validate protein structures
Authors: Vila Jorge A. , Scheraga Harold A. 
Published: November 2007

Interest centers here on the analysis of two different, but related, phenomena that affect side‐chain conformations and consequently 13Cα chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13Cα chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13Cα chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13Cα chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2ξ possible ionization states of the whole molecule, with ξ being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshielding of the 13Cα nucleus, indicated that: (i) there is a significant difference in the computed 13Cα chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13Cα nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13Cα chemical shifts of a protein in solution. The second is how side‐chain flexibility influences computed 13Cα chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR‐derived structure with the backbone conformation assumed to be fixed. The 13Cα chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side‐chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side‐chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13Cα chemical shifts (by up to 3.7 ppm), was found for ∼68% and ∼63% of all ionizable residues and all non‐Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root‐mean‐square‐deviation (1.97 ppm ≤ rmsd ≤ 2.13 ppm), between computed and observed 13Cα chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13Cα chemical shifts and χ1 torsional angles (given by the vicinal coupling constants, 3JN−Cγ and 3JC′−Cγ, is discussed. Proteins 2008. © 2007 Wiley‐Liss, Inc.