13C NMR reveals no evidence of n-π* interactions in proteins

Abstract

An n = π* interaction between neighboring carbonyl groups has been postulated to stabilize protein structures. Such an interaction would affect the (13)C chemical shielding of the carbonyl groups, whose paramagnetic component is dominated by n = π* and π = π* excitations. Model compound calculations indicate that both the interaction energetics and the chemical shielding of the carbonyl group are instead dominated by a classical dipole-dipole interaction. A set of high-resolution protein structures with associated carbonyl (13)C chemical shift assignments verifies this correlation and provides no evidence for an inter-carbonyl n = π* interaction.

Figure 1. Predicted Interaction and Associated Carbonyl C Chemical Shifts.

(A) Residues Asn155 and Phe189 from the x-ray structure of Bacillus amyloliquefaciens subtillisin BPN’ (PDB ID: 1v5i) illustrating the structural features for an optimal interaction between carbonyl groups. (B) 2D contour plot of carbonyl C chemical shift differences relative to random coil values as a function of the distance () and angle () between carbonyls. A Gaussian smoothing function was applied to the data with and of 0.3 Å and 1.5°, respectively. A transparency mask based on the density of experimental data (see also Figure S1) is overlaid on the contour plot. Regions lacking experimental data are white. Positive values indicate downfield shifts.

Figure 2. Carbonyl C Chemical Shifts and Dipole-Dipole Potential.

Carbonyl C chemical shift differences relative to random coil are plotted against calculated dipole-dipole potential (). The dipole-dipole potential is calculated from the high-resolution x-ray structure using Equation 1. Pairs of carbonyls with and values within the optimal limits for an interaction are colored red.

Figure 3. Carbonyl C Chemical Shifts and Hydrogen Bonds.

Contour plot of C carbonyl chemical shift differences as a function of calculated dipole-dipole potential () and calculated hydrogen bond length (). See also Figure S2.

Figure 4. Formamide Trimer Model.

Molecular orbitals of (A) the hydrogen bond donor, (B) the putative donor and (C) the putative acceptor, in the trimeric complex used in quantum chemical calculations.

Figure 5. Summary of Quantum Chemical Calculations.

Plot of calculated (A) carbonyl C chemical shielding () and (B) dipole-dipole interaction energy () as a function of the distance between donor oxygen and acceptor carbon () and the angle between carbonyl groups (). See also Figure S3.

Figure 6. Summary of Quantum Chemical Calculations for ‘End-On’ Dipole Interaction.

Plots of (A) interaction energy and (B) carbonyl C chemical shielding () as a function of the angle between the carbonyls () for three different distances () between the donor oxygen and acceptor carbon.