Anomalous Amide Proton Chemical Shifts as Signatures of Hydrogen Bonding to Aromatic Sidechains

Hydrogen bonding between an amide group and the p-π cloud of an aromatic ring was first identified in a protein in the 1980s. Subsequent surveys of high-resolution X-ray crystal structures found multiple instances, but their preponderance was determined to be infrequent. Hydrogen atoms participating in a hydrogen bond to the p-π cloud of an aromatic ring are expected to experience an upfield chemical shift arising from a shielding ring current shift. We survey the Biological Magnetic Resonance Data Bank for amide hydrogens exhibiting unusual shifts as well as corroborating nuclear Overhauser effects between the amide protons and ring protons. We find evidence that Trp residues are more likely to be involved in p-π hydrogen bonds than other aromatic amino acids, whereas His residues are more likely to be involved in hydrogen bonds with a ring nitrogen acting as the hydrogen acceptor. The p-π hydrogen bonds may be more abundant than previously believed. The inclusion in NMR structure refinement protocols of shift effects in amide protons from aromatic side chains, or explicit hydrogen bond restraints between amides and aromatic rings, could improve the local accuracy of side-chain orientations in solution NMR protein structures, but their impact on global accuracy is likely be limited.

Amide proton transfer imaging for differentiation of tuberculomas from high-grade gliomas: Preliminary experience

Purpose Tuberculomas can occasionally masquerade as high-grade gliomas (HGG). Evidence from magnetisation transfer (MT) imaging suggests that there is lower protein content in the tuberculoma microenvironment. Building on the principles of chemical exchange saturation transfer and MT, amide proton transfer (APT) imaging generates tissue contrast as a function of the mobile amide protons in tissue’s native peptides and intracellular proteins. This study aimed to further the understanding of tuberculomas using APT and to compare it with HGG. Method Twenty-two patients ( n = 8 tuberculoma; n = 14 HGG) were included in the study. APT was a 3D turbo spin-echo Dixon sequence with inbuilt B0 correction. A two-second, 2 μT saturation pulse alternating over transmit channels was applied at ±3.5 ppm around water resonance. The APT-weighted image (APTw) was computed as the MT ratio asymmetry (MTRasym) at 3.5 ppm. Mean MTRasym values in regions of interest (areas = 9 mm2; positioned in component with homogeneous enhancement/least apparent diffusion coefficient) were used for the analysis. Results MTRasym values of tuberculomas ( n = 14; 8 cases) ranged from 1.34% to 3.11% ( M = 2.32 ± 0.50). HGG ( n = 17;14 cases) showed MTRasym ranging from 2.40% to 5.70% ( M = 4.32 ± 0.84). The inter-group difference in MTRasym was statistically significant ( p < 0.001). APTw images in tuberculomas were notable for high MTRasym values in the perilesional oedematous-appearing parenchyma (compared to contralateral white matter; p < 0.001). Conclusion Tuberculomas demonstrate lower MTRasym ratios compared to HGG, reflective of a relative paucity of mobile amide protons in the ambient microenvironment. Elevated MTRasym values in perilesional parenchyma in tuberculomas are a unique observation that may be a clue to the inflammatory milieu.

Alternating handedness motifs in proteins classify structure and cofactor binding

Cofactor binding sites in proteins often are composed of favorable interactions of specific cofactors with the sidechains and/or backbone protein fold motifs. In many cases these motifs contain left-handed conformations which enable tight turns of the backbone that present backbone amide protons in direct interactions with cofactors termed ‘cationic nests’. Here, we defined alternating handedness of secondary structure as a search constraint within the PDB to systematically identify these cofactor binding nests. We identify unique alternating handedness structural motifs which are specific to the cofactors they bind. These motifs can guide the design of engineered folds that utilize specific cofactors and also enable us to gain a deeper insight into the evolution of the structure of cofactor binding sites.

Direct and Indirect Models for Protein Chemical Denaturation are Characteristic of Opposite Dynamic Properties

Two major models, namely direct and indirect models, have been proposed for the protein chemical denaturation but it remains challenging to experimentally demonstrate and distinguish between them. Here, by use of CD and NMR spectroscopy, we succeeded in differentiating the effects on a small but well-folded protein WW4, of GdmCl and NaSCN at diluted concentrations (≥200 mM). Both denaturants up to 200 mM have no alternation of its average structure but do reduce its thermodynamic stability to different degrees. Despite acting as the stronger denaturant, GdmCl only weakly interacts with amide protons, while NaSCN shows extensive interactions with both hydrophobic side chains and amide protons. Although both denaturants show no significant perturbation on overall ps-ns backbone dynamics of WW4, GdmCl suppresses while NaSCN enhances its μs-ms backbone dynamics in a denaturant concentration dependent manner. Quantitative analysis reveals that although they dramatically raise exchange rates, GdmCl slightly increases while NaSCN reduces the population of the major conformational state. Our study represents the first report deciphering that GdmCl and NaSCN appear to destabilize a protein following two models respectively, which are characteristic of opposite μs-ms dynamics.