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. Author manuscript; available in PMC: 2020 Aug 20.
Published in final edited form as: Biochemistry. 2019 Aug 6;58(33):3480–3493. doi: 10.1021/acs.biochem.9b00355

Figure 3. Stereospecific NMR assignment of asparagine NH2 side chain protons.

Figure 3.

(A) Wild-type pp32 NHz-filtered HSQC spectrum. Ladder side chain NH resonances are connected by dashed lines. For N98, only the upfield resonance is resolved, though the position of the downfield proton is known from HDX experiments where overlapping peaks exchange rapidly (Figure S7). (B) Schematic diagram of an E.COSY cross-peak pattern with a large positive J(A-X) coupling and a small positive J(B-X) coupling (brackets). Solid circles (black) represent observed resonances in which A/B nuclei are paired with the same X spin state (here, Xα) whereas dashed circles (grey) represent unobserved resonances in which A/B nuclei are paired with opposite X states (indicated by labels next to peaks). When J(A-X) and J(B-X) coupling constants have the same sign, the line connecting the observed resonances has a positive slope, as shown. J(A-X) and J(B-X) coupling constants of opposite sign have negative slope. (C) Selections from C123N CγO-coupled NH2-HSQC E.COSY spectra to measure small 2J(HE/Z-Cγ) values. 2J(HE-Cγ) is positive (1 to 5 Hz, same as (B)) whereas 2J(HZ-Cγ) is negative (−1 to −5 Hz), permitting the Hz and HE resonances to be assigned using the large positive 1J(Nδ2-Cγ) coupling constant. For the ladder asparagines 74 and 98, the upfield resonances are displaced with positive slope (as in B), identifying these resonances as originating from HE. The downfield resonances are displaced with negative slope, identifying these resonances as originating from Hz. For non-ladder asparagine residues such as N122 (right panel), this pattern is reversed. The full spectrum is shown in Figure S5. (D) Selections from T49L HNCO E.COSY to measure small 3J (HE/Z-Cβ) values. 3J(Hz-Cβ) is positive (5–10 Hz) whereas 3J(Hz-Cβ) is close to zero, permitting the Hz and HE resonances to be assigned using the large positive 1J(Cγ-Cβ) coupling constant. For the ladder asparagines, downfield resonances are displaced with a positive slope, identifying these resonances as originating from HZ, whereas upfield resonances have a vertical displacement, identifying these resonances as originating from HE. Again, for non-ladder asparagine side chains (e.g., N122, right panel), this pattern is reversed. The full spectrum shown in Figure S5.