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

摘要

In this study, the configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein are determined via measuring differential and across-hydrogen-bond H/D isotope effects by the IS-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl ¹³C′ shifts, defined as Δ²Δ¹³C′ (ND) = ²Δ¹³C′ (ND^E) − ²Δ¹³C′ (ND^Z), provide a reliable means for the configuration assignment for side-chain amides, as environmental effects (hydrogen bonds and charges etc.) are greatly attenuated over the two bonds separating 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, defined as Δ¹Δ¹⁵N(D) = ¹Δ¹⁵N(D^E) − ¹Δ¹⁵N(D^Z), 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 across-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks involving 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. The rotamer correction via differential isotope effects is not only robust but simple and can be applied to large proteins.