1. Protein
NMR
Part II

2. 1. Protein Structures by NMR
NMR, UNLIKE Xray crystallography
and EM, DOES NOT experimentally
produce a protein structure.
NMR yields Distance Restraints,
which are used to CALCULATE
protein structures.
NMR structure calculations yield
MULTIPLE solutions (ensembles).
NMR protein structures are
always ENSEMBLE AVERAGES.

3. 2. NMR Structure Calculation
H3 Lys10
H2 Arg98 > Int. 0.75
H1 Ser57
H2 Glu42 > Int. 0.45
H2 Asp11
H2 Arg98 > Int. 0.55
H3 Lys72
H2 Arg98 > Int. 0.95
Long-Range/Short-Range
Distance Restraints
Energy Minimization
Short-Range > 2nd. Structure
Long-Range > Tertiary Structure
(Protein Fold)
Assessment of Structural Quality
r.m.s.d., Ramachandran Plot etc.

4. 3. The Nuclear Overhauser Effect (NOE Experiments)
Proton-Proton Distances

5. 4. Problems with Proteins
Spectral Overlap
Spectral Editing (2D or 3D) 1H 1H
15N
Selective Labeling

6. 5. Protein NOEs
-helical NOEs
-sheet NOEs

7. 6. … yet, how do we know which one is which?
Goal: Identify the Resonance
Frequencies of ALL Proton,
Carbon and Nitrogen nuclei
in a protein.
Backbone Assignment Strategies
Side-Chain Assignment Strategies

8. 7. Magnetization Transfer
Magnetization transfer through space > NOE
Magnetization transfer through bonds > J-coupling
1H/15N Correlation (2D)
HSQC or HMQC-type i
i (-1)
HNCA Experiment (3D)
HNCACB Experiment (3D)
15N 1H

9. 8. Assignment HNCACB Experiment (as an example) What have we learned?
C i
C i (-1)
HNCACB Experiment
(as an example) i
i (-1)
C i
C i (-1)
What have we learned?
The Amide Proton resonance frequency
The Amide Nitrogen resonance frequency
The Alpha Carbon resonance frequency
The Beta Carbon resonance frequency
i (-1) i

10. 9. NMR Experiments Types of experiments and nomenclature
BACKBONE EXPERIMENTS
HNCA
HN(CO)CA
HN(CA)CO
HNCO
HNCACB
HN(CO)CACB

11. 9. NMR Experiments (cont.)
Types of experiments and nomenclature
SIDE-CHAIN EXPERIMENTS
(H)CCNH
(H)CC(CO)NH
H(CC)NH
H(CC)(CO)NH
HCCH-COSY
HCCH-TOCSY

12. 10. Putting things together …
Hence: A 3D HCCH-TOCSY
looks like a
3D 13C-edited NOESY
without NOE Cross-peaks.

13. 11. NMR Pulse-Sequences How do we READ them?
What do they MEAN and how do they WORK?

14. 12. Let’s do one together …
Can you point out the basic BUILDING BLOCKS?
Can you determine the MAGNETIZATION TRANSFER PATH?
Can you identify the NMR EXPERIMENT?

15. 13. Summary
NMR structures are COMPUTED and not experimentally determined.
To calculate an NMR structure we need DISTANCE RESTRAINTS.
Distance restraints are provided by NOE EXPERIMENTS.
NOE experiments are SPECTRAL EDITED and recorded in 3D.
NOE experiments are meaningless unless we have a
COMPLETE NMR RESONANCE ASSIGNMENT.
Resonance assignments are given by BACKBONE- and SIDE-CHAIN
NMR EXPERIMENTS (which exploit J-COUPLINGS).
Backbone- and side-chain experiments are recorded as sets of
3D EXPERIMENTS employing tailored NMR PULSE SEQUENCES.
NMR pulse-sequences are made up of specifically arranged
BUILDING BLOCKS .