Hncacb Assignment Sheet

Reference:
S. Grzesiek and A. Bax (1992) J. Magn. Reson.99 201-207. (Link to Article)

Minimum labelling:15N, 13C

Dimensions: 3

Magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to 15NH and then to 1HN for detection. Transfer form Cαi-1 can occur both to 15Ni-1 and 15Ni, or viewed the other way, magnetisation is transferred to 15Ni from both 13i and 13i-1. Thus for each NH group there are two Cα and Cβ peaks visible. The chemical shift is evolved simultaneously on 13Cα and 13Cβ, so these appear in one dimension. The chemical shifts evolved in the other two dimensions are 15NH and 1HN.

Along with the CBCA(CO)NNH and HSQC this forms the standard set of experiments needed for backbone assignment. For large proteins the signal-to-noise may not be great and assignment using the HNCA, HN(CO)CA, HNCO and HN(CA)CO may form a better strategy. When using deuterated protein, the spectrum has to be recorded as an ‘out-and-back’ method and the signal-to-noise suffers even further.

Residues of DNA polymerase β (β-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/13C/2H/1H,13C-methylLeu,Ile,Val-labeled β-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A ΔV303–V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of β-Pol contacts an edge/face region of the β sheet of the XRCC1 NTD, while the β-Pol palm weakly contacts the α2 helix.

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