We have investigated the carbon-13 solution nuclear magnetic resonance (NMR) chemical shifts of Cα, Cβ, and Cγ carbons of 19 valine residues in a vertebrate calmodulin, a nuclease from Staphylococcus aureus, and a ubiquitin. Using empirical chemical shift surfaces to predict Cα, Cβ shifts from known, X-ray φ,ψ values, we find moderate accord between prediction and experiment. Ab initio calculations with coupled Hartree−Fock (HF) methods and X-ray structures yield poor agreement with experiment. There is an improvement in the ab initio results when the side chain χ1 torsion angles are adjusted to their lowest energy conformers, using either ab initio quantum chemical or empirical methods, and a further small improvement when the effects of peptide-backbone charge fields are introduced. However, although the theoretical and experimental results are highly correlated (R 2 ∼ 0.90), the observed slopes of ∼−0.6−0.8 are less than the ideal value of −1, even when large uniform basis sets are used. Use of density functional theory (DFT) methods improves the quality of the predictions for both Cα (slope = −1.1, R 2 = 0.91) and Cβ (slope = −0.93, R 2 = 0.89), as well as giving moderately good results for Cγ. This effect is thought to arise from a small, conformationally-sensitive contribution to shielding arising from electron correlation. Additional shielding calculations on model compounds reveal similar effects. Results for valine residues in interleukin-1β are less highly correlated, possibly due to larger crystal−solution structural differences. When taken together, these results for 19 valine residues in 3 proteins indicate that choosing the lowest energy χ1 conformer together with X-ray φ,ψ values enables the successful prediction of both Cα and Cβ shifts, with DFT giving close to ideal slopes and R 2 values between theory and experiment. These results strongly suggest that the most highly populated valine side-chain conformers are those having the lowest (computationally determined) energy, as evidenced by the ability to predict essentially all Cα, Cβ chemical shifts in calmodulin, SNase, and ubiquitin, as well as moderate accord for Cγ. These observations suggest a role for chemical shifts and energy minimization/geometry optimization in the refinement of protein structures in solution, and potentially in the solid state as well.

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Using gauge-including atomic orbital self-consistent field ab initio quantum chemical methods, we have computed the effects of bond lengths, bond angles, and torsion angles on the carbon-13 chemical shielding of Ca (and C@) sites in model fragments for glycine, alanine, and valine residues in proteins. Predicted chemical shieldings are highly sensitive to bond length variations, and we show that it is essential to relax or energy minimize protein structures (to remove large errors associated with bond length uncertainties) in order to successfully predict experimental l3C N M R spectra.

We report the 470-MHz (11.7 T) 19F solution nuclear magnetic resonance (NMR) spectra of 2-, 3-, and 4-fluorophenylalanine incorporated into the egg white lysozymes (EC 3.2.1.17) of chicken, pheasant, and duck, as well as spectra of 4-fluorotryptophan incorporated into chicken, California valley quail, and Bob White quail and 5- and 6-fluorotryptophan-labeled chicken lysozyme. The 19F solution NMR spectrum of [4-F]Phe hen egg white lysozyme (HEWL) consists of three sharp resonances, which span a total chemical shift range of 4.8 ppm (at p2H = 6.1). For [3-F]Phe HEWL, the chemical shift range is much smaller, 1.0 ppm (at p2H = 5.9), due presumably to the occurrence of fast phenyl ring flips about the C beta-C gamma bond axis. For [2-F]Phe HEWL, six resonances are observed, spanning a chemical shift range of 7.4 ppm (at p2H = 5.8), due to slow C beta-C gamma ring flips, i.e., both ring-flip isomers appear to be "frozen in" because of steric hindrance. Rotation of the [2-F]Phe residues remains slow up to 55 degrees C (p2H = 4.7). With the [F]Trp-labeled proteins, we find a maximal 14.6-ppm shielding range for [4-F]Trp HEWL but only a 2.8- and 2.4-ppm range for [5- and 6-F]Trp HEWL, respectively, due presumably to increased solvent exposure in the latter cases. Guanidinium chloride denaturation causes loss of essentially all chemical shift nonequivalence, as does thermal denaturation. Spectra recorded as a function of pH show relatively small chemical shift changes (< 1.4 ppm) over the pH range of approximately 1.2-7.8.(ABSTRACT TRUNCATED AT 250 WORDS)