Aizhuo Liu

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′(ND^E)‐²Δ¹³C′(ND^Z), 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(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 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.

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.