Hydrogen‐Bond Detection, Configuration Assignment and Rotamer Correction of Side‐Chain Amides in Large Proteins by NMR Spectroscopy through Protium/Deuterium Isotope Effects

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Abstract
The configuration and hydrogen‐bonding network of side‐chain amides in a 35 kDa protein were determined by measuring differential and trans‐hydrogen‐bond H/D isotope effects by using the isotopomer‐selective (IS)‐TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two‐bond isotope effects on carbonyl 13C′ shifts, which are defined as Δ2Δ13C′(ND)=2Δ13C′(NDE)‐2Δ13C′(NDZ), provide a reliable means for the configuration assignment for side‐chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one‐bond isotope effects on 15N chemical shifts, which are defined as Δ1Δ15N(D)=1Δ15N(DE)‐1Δ15N(DZ) can be correlated with hydrogen‐bonding interactions, particularly those involving charged acceptors. The differential one‐bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H‐bonding interactions, and charge effects. Furthermore, the pattern of trans‐H‐bond H/D isotope effects can be mapped onto more complicated hydrogen‐bonding networks that involve bifurcated hydrogen‐bonds. Third, the correlations between Δ1Δ15N(D) and hydrogen‐bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side‐chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.

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