Hncacb Assignments

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.

Introduction

The interpretation and assignment of NMR data of a protein usually starts with assignment of the backbone atoms and linking them with their sequential neighbours. The experiments that we will use at this stage are:

  • 2D 15N-HSQC:  Provides an initial set of HN resonances that will be used as 'root' resonances
  • CBCAcoNH: provides CB and CA resonances of residue i-1 and HN resonances of residue i
  • HNCACB: provides CB and CA resonances of residues i,i-1 and HN resonances of residue i
  • HBHAcbcacoNH: provides HB and HA resonances of residue i-1 and HN resonances of residue i
  • HNcaHA: provides HA resonances of residues i,i-1 and HN resonances of residue i
  • hCCH-TOCSY: provides side chain resonances starting from HA, CA, HB and CB resonances
These experiments allow us to assign most of the HA, HB, CA, CB and HN resonances of many residues in NapD and group them into spin systems. The CA and CB chemical shifts can be used to make an initial score for the type of residue that a particular spin system belongs to, which in combination with sequential contacts can provide part of the sequential assignment. However, spin system typing is much more accurate when more side chain assignments are available and help the identification of stretches of sequentially linked residues by comparing the sequentially linked spin system types to the primary sequence of the protein.

Schematic drawings of the atoms detected in the backbone assignment experiments and the patterns that can be found in the corresponding 3D spectra, are depicted on the right.

Strategy

In short the strategy that we will follow here is:

  • 15N-HSQC peak picking.
  • Initialising of root resonances and spin systems based on the 15N-HSQC.
  • Linking the root resonances to 3D spectra; picking 3D peaks and transferring the root resonance assignments to the 3D peaks.
  • Assignment of resonances to HA, CB, HA, HB atom types
  • Connecting backbone resonances to side chain resonances
  • Assigning spin systems to residue types
  • Linking residues that exhibit sequential connectivities in the NMR spectra

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