In this work, we functionalized a polyester backbone via sequential Huisgen 1,3-dipolar cycloaddition and 3-component Passerini reactions.
By docking capsaicin to the closed state, or to open states with or without backbone movements, the sequential events underlying capsaicin binding and activating TRPV1 started to emerge.
For very large proteins in the highest magnetic fields, the large chemical shift anisotropy (CSA) of carbonyl carbon deteriorates coherence transfer efficiency in experiments designed for unambiguous sequential backbone assignment.
Hence, a suite of triple-resonance NMR experiments was conducted on [U-H, N, C]-Fe3+-HO2O for sequential backbone assignments.
Because we suspected conformational disorder was hindering crystal growth, we turned to solution NMR and obtained the sequential backbone assignments of wt, Y75A, and H83A apo-HasAp.
Sequential backbone chemical shift assignments (H, N, and Cα) have been deposited in the BMRB as entries 19720 (wt apo-HasAp), 19721 (Y75apo-HasApand, and 19722 (H83apo-HasApAp).
A suite of five experiments (see Experimental Procedures) allowed sequential backbone assignments for 93% of non-proline residues in wt apo-HasAp (Table S1 of the Supporting Information).
Sequential backbone assignments of the U-N,C-labeled DC-SIGN CRD (500 μM) were made at 37 °C using three-dimensional intra- and inter-residue coherence transfer experiments, HNCA, HNCB (HNCACB with tauCC = 7 ms), and HN CO CACB.
The spectra recorded for sequential backbone assignments included the TROSY versions of 2D N-H HSQC and 3D HNCO, HN CA CO, HNCA, HN CO CA, HNCACB, and HN CO CACB TROSY experiments.
Approximately 1.2 mM Fe3+-HO2O and 0.67 mM Fe3+-HO2R [U-H, N, C]-HO2 in 50 mM Tris, 50 mM KCl pH 7.0 plus 10% H2O were used to conduct triple-resonance experiments for sequential backbone assignments.
