Protein
NMR
Part II
1. Protein Structures by NMR
NMR, UNLIKE Xray crystallograph
and EM, DOES NOT experimenta
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.
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.
3. The Nuclear Overhauser Effect
(NOE Experiments)
Proton-Proton Distances
4. Problems with Proteins
Spectral Overlap
Spectral Editing (2D or 3D)
Selective Labeling
1
1
H
H
15N
5. Protein NOEs
-sheet NOEs
-helical
NOEs
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
7. Magnetization Transfer
Magnetization transfer through space > NOE
Magnetization transfer through bonds > J-coupling
i
1H/15N
Correlation (2D)
HSQC or HMQCtype
HNCA Experiment (3D)
HNCACB Experiment (3D)
15N
i (-1)
1H
8. Assignment
C i
C i (-1)
HNCACB Experiment
(as an example)
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)
C i
i (-1) i
9. NMR Experiments
Types of experiments and nomenclature
BACKBONE EXPERIMENTS
HNC
A
HN(CA)C
O
HNCAC
B
HN(CO)CA
HNCO
HN(CO)CAC
B
9. NMR Experiments (cont.)
Types of experiments and nomenclature
SIDE-CHAIN EXPERIMENTS
(H)CCN
H(CC)N
H
H
HCCHCOSY
(H)CC(CO)NH
H(CC)(CO)NH
HCCH-TOCSY
10. Putting things together …
Hence: A 3D HCCH-TOCSY
looks like a
3D 13C-edited NOESY
without NOE Cross-peaks.
11. NMR Pulse-Sequences
How do we READ them?
What do they MEAN and how do they
WORK?
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?
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 SIDECHAIN
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 .