Proton 1D spectrum with water suppression

Sample: 1mM N15 labeled wild type ubiquitin, MW 8.5 kDa, pH6.8

1H-15N SOFAST HMQC

Sample: N15 labeled DNA damage-inducible protein 1 (MW 47kDa), 70 uM, pH6.8

1H-13C HSQC

Sample: Amino acid mix (unlabeled), 1mM concentration each, at pH6.8

2D TOCSY

2D TOCSY spectrum provides information of proton signals of amino acids in a protein (spin systems). Assigning those signals is a preparation step of structure determination by NMR. To successfully assign all the spin systems in TOCSY, 2D-HSQC and 2D-NOESY are also required to help identifying residue types and establishing residue connectivity.

 

 

 

Sample: UBA domain of Ddi1 protein 1mM, unlabeled, pH 6.8

2D NOESY

2D NOESY experiment provides NOE information of protons close in space, and the NOE signal intensity is related to the distance between two protons. Therefore, the NOESY spectrum may provide proton three-dimensional network information of an organic compound, a protein or an nucleic acid, which is very useful for structure determination.

 

Sample: protein 1mM, unlabeled, pH 6.8

 

Details:

 

 

NMR titration

In this example, N15-labeled UIM motif of proteasome subunit Rpn10 was titrated with three different proteins(ligands), ubiquitin, diubiquitin, and the UBL domain of Dsk2. The figure below demonstrated signal migration of several Rpn10 amide protons upon ligand binding.

 

Based on overall chemical shift changes of Rpn10 amide proton signals, the binding interface on Rpn10-UIM can be identified.

 

Furthermore, by measuring signal chemical shift change at each titration point, the titration curve of individual residues can be plotted out, and binding affinity Kd can be calculated sequentially.

Coming soon!