NMR Flashcards
NMR fundamentals
Nucleic has quantum number (I = 1/2) will give signal in NMR (1H, 13C, 15N, 31P) I = 1/2 gives to states
Coherence
Magnetization transfer between nuclear neighbors.
Coherence transfer gives information on which nuclei are covalently bonded.
COSY
Direct neighbors
TOCSY
Entire spin - coupled system ¨spin-systems¨
NOESY
The cross of a NOESY spectrum indicate which protons are close to each other in space.
Basic strategy for NMR assignment of proteins
1, identify spin system for each amino acid
2, put them in order: sequence specific assignment
Spinn relaxation (nOe)
Magnetization transfer through space - distance dependent.
Required for put the spin system in order.
Sequential NOE
Internal NOE
2D NMR NOESY spectrum
Depicts intermolecular distances between all protons in the biomolecule.
Magnetic labelling
Requires gene technology and bacterial over expression
15N labelling
HSQC
Each peak corresponds to one amino acid
3D NOESY-HSQC
HSQC experiment can be recorded and analyzed separately
13C - labelling
With this we can do the sequential assignment by through-bond connectivities ¨Backbone experiments¨
side chain assignments are required for structure determination by NoEs
Calculating an NMR structure requires
Basics
protein sequence, covalent structure
assignments of all relevant NMR resonances
Conformational constraints NoEs (between H) Hydrogen bonds Dihedral angles Chemical shifts Residual dipolar couplings
Secondary structure elements can be identified by unique short distances
α-helix
(CαH)i - NHi+3
(CαH)i - (CβH) i+3
β-sheets
NH-NH across the sheet
CαH - CαH across the sheet
CαHi - NHi+1 along the chain
problem at higher molecular weights
Slow movement in solution makes signal relax faster poor signal-to-noise and line broadening.
interaction between relaxing nuclei increases relaxation
Overlap also in 2D and 3D spectra