Introduction to protein backbone assignment

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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