NMR detection and conformational dependence of two, three, and four-bond isotope shifts due to deuteration of backbone amides

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NMR detection and conformational dependence of two, three, and four-bond isotope shifts due to deuteration of backbone amides

Abstract

NMR isotope shifts occur due to small differences in nuclear shielding when nearby atoms are different isotopes. For molecules dissolved in 1:1 H2O:D2O, the resulting mixture of N-H and N-D isotopes leads to a small splitting of resonances from adjacent nuclei. We used multidimensional NMR to measure isotope shifts for the proteins CUS-3iD and CspA. We observed four-bond 4â??N(ND) isotope shifts in high-resolution 2D 15N-TROSY experiments of the perdeuterated proteins that correlate with the torsional angle psi. Three-bond 3â??Câ??(ND) isotope shifts detected in H(N)CO spectra correlate with the intraresidue H-O distance, and to a lesser extent with the dihedral angle phi. The conformational dependence of the isotope shifts agree with those previously reported in the literature. Both the 4â??N(ND) and 3â??Câ??(ND) isotope shifts are sensitive to distances between the atoms giving rise to the isotope shifts and the atoms experiencing the splitting, however, these distances are strongly correlated with backbone dihedral angles making it difficult to resolve distance from stereochemical contributions to the isotope shift. H(NCA)CO spectra were used to measure two-bond 2â??Câ??(ND) isotope shifts and [D]/[H] fractionation factors. Neither parameter showed significant differences for hydrogen-bonded sites, or changes over a 25° temperature range, suggesting they are not sensitive to hydrogen bonding. Finally, the quartet that arises from the combination of 2â??Câ??(ND) and 3â??Câ??(ND) isotope shifts in H(CA)CO spectra was used to measure synchronized hydrogen exchange for the sequence neighbors A315-S316 in the protein CUS-3iD. In many of our experiments we observed minor resonances due to the 10% D2O used for the sample deuterium lock, indicating isotope shifts can be a source of spectral heterogeneity in standard NMR experiments. We suggest that applications ofÂ*isotope shifts such as conformational analysis and correlated hydrogen exchange could benefit from the larger magnetic fields becoming available.



Source: Journal of Biomolecular NMR