3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

Abstract

The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-(2)H,(15)N,(13)C; Ileδ1-{(13)CH(3)}; Leu,Val-{(13)CH(3)/(12)CD(3)}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-ΔYRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40 °C to improve the sensitivity of the measurements and alleviate the overlap of (1)H-(15)N correlations in the abundant α-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of (1)H-(15)N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D (1)H-(15)N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected 'out-and-back' HMCM(CG)CBCA experiment, and (3) strong sequential HN-HN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578-195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.

Cooperative dynamics across distinct structural elements regulate PTP1B activity

“…All PTP1B 13 C-methyl ILV assignment NMR measurements were performed using a Bruker Advance Neo 800 MHz NMR spectrometer with a TCI HCN-active z-gradient cryoprobe at 298 K. A 3D ( 13 C, 13 C, 1 H) HMCM(CG)CBCA experiment functioned as the initial assignment step using PTP1B produced following scheme 1 (32). A 3D ( 13 C, 13 C, 1 H) HSQC-NOESY-HSQC (τm= 200 ms) and a 3D ( 13 C, 15 N, 1 H) HSQC-NOESY-HMQC (τm = 400 ms) spectra were recorded using PTP1B produced following scheme 3.…”

Heteronuclear NMR studies on 44 kDa dimer, syndesmos

“…Therefore, deuterium labeling method was carried out to overcome low signal-to-noise and resolution by increasing relaxation rate. [10][11][12][13][14] Based on a series of triple resonance spectra measured on a triple isotope-[ 2 H/ 13 C/ 15 N]-labeled protein, TROSY-based triple resonance spectra {HNCACB, HNCA and HNCO} [15][16][17] were obtained showing well dispersed and homogeneous signals. Here, we report the sequence-specific backbone resonance assignments of syndesmos partially, but assigned NMR data in combination with structural information can be used for the studies on interaction with cell adhesion adaptor protein including syn4 cyto in the future.…”

Divide and conquer is always best: sensitivity of methyl correlation experiments

“…One of the key experiments developed in that work is the HMCM[CG]CBCA pulse sequence, which simultaneously correlates the methyl carbon and proton shifts to the rest of the carbons on the sidechain in a single spectrum. The HMCM[CG]CBCA experiment has been successfully used to assign methyl resonances in a number of smaller proteins (Wang et al, 2012; Krejcirikova and Tugarinov, 2012; Zhuraviena et al, 2012; Chan et al, 2012). In the case of larger systems, this experiment has been replaced by a “divide-and-conquer” strategy, whereby the shifts of the individual carbons, e.g.…”