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:Â 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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Introduction
Some NMR spectra for sequential backbone assignment
Observable nuclei are indicated in colors, which depend on the sign of the peak:
blue: positive
purple: negative
The nuclei corresponding to the NH root frequencies are colored in green.
For illustration purposes, dashed circles are used to depict virtual peaks positions.
Combination of HNCACB and CBCAcoNH, allows to distinguish between sequential (i-1) and intra residual (i) peaks, and between CA and CB resonances.
Combination of HNcaHA and HBHAcbcacoNH, allows to distinguish between sequential (i-1) and intra residual (i) peaks, and between HA and HB resonances.
