NMR Flashcards
Chemical shifts and structure
If shift is higher than random coil -> sheet
If lower -> helix
Major and minor contributions
Environment
Secondary structure
Isotopic enrichment
90% minimal media
13C glucose or 15N ammonium sulphate
Which residues show in a 15N HSQC?
Side chain NH- e.g. R and D, but not NH3s
Histidine and Pro not seen
Extra peak for N terminus if it is visible (NH3)
Need to minus for C terminus?
Kd value
Systemic titration of ligand mapped against shift
MW and NMR
Make tumble quicker
Time through 57.3 = correlation time Stokes Einstein relationship Radius increases, TM increases Makes the lines broad Heating- lowers viscosity and increases tumble rate
TROSY
Triple labelled 13C, 15N, 2H Deuteration reduces broadening Transverse relaxation optimised spectroscopy Only non labile are converted
Why do NH3 not show
High rate of atomic exchange with water
NH is much slower so can be seen but is broad
Two types of correlation experiments
Through bond and through space
2 ways to monitor ligand interactions
HSQC- mgs of protein and 15N
Or changes in 1H of ligand when add protein
STD NMR
RF on RF off Difference specta Blank = no binding Binding gives peaks
WATER LOGSY
Water ligand gradient observed by gradient spectroscopy
Protein saturates water around active site
Water transfer signal to ligand
Binding- positive
No binding- negative
But- blank can mean binding
