Protein NMR Part II. 1. Protein Structures by NMR NMR, UNLIKE Xray crystallography and EM, DOES NOT...
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Transcript of Protein NMR Part II. 1. Protein Structures by NMR NMR, UNLIKE Xray crystallography and EM, DOES NOT...
ProteinNMRPart II
1. Protein Structures by NMR NMR, UNLIKE Xray crystallography
and EM, DOES NOT experimentallyproduce a protein structure.
NMR yields Distance Restraints,which are used to CALCULATEprotein structures.
NMR protein structures arealways ENSEMBLE AVERAGES.
NMR structure calculations yieldMULTIPLE solutions (ensembles).
2. NMR Structure Calculation H3 Lys10 H2 Arg98 > Int.
0.75H1 Ser57 H2 Glu42 > Int.
0.45H2 Asp11 H2 Arg98 > Int.
0.55H3 Lys72 H2 Arg98 > Int.
0.95
Long-Range/Short-RangeDistance Restraints
Short-Range > 2nd. StructureLong-Range > Tertiary Structure
(Protein Fold)
Energy Minimization
Assessment of Structural Qualityr.m.s.d., Ramachandran Plot etc.
3. The Nuclear Overhauser Effect (NOE Experiments)
Proton-Proton Distances
4. Problems with Proteins
Spectral Editing (2D or 3D)1H 1H1H 1H 15N
Selective Labeling
Spectral Overlap
5. Protein NOEs-sheet NOEs-helical NOEs
6. … yet, how do we know which one is which?
Goal: Identify the ResonanceFrequencies of ALL Proton,Carbon and Nitrogen nucleiin a protein.
Backbone Assignment StrategiesSide-Chain Assignment Strategies
7. Magnetization Transfer Magnetization transfer through space >
NOEMagnetization transfer through bonds > J-coupling
i
i (-1)
1H/15N Correlation (2D)HSQC or HMQC-type
15N
1H
HNCA Experiment (3D)HNCACB Experiment
(3D)
8. Assignment
ii (-1)
C i
C i (-1)
C i
C i (-1)
HNCACB Experiment(as an
example)What have we
learned?The Amide Proton resonance frequencyThe Amide Nitrogen resonance frequencyThe Alpha Carbon resonance frequencyThe Beta Carbon resonance frequency
i
i (-1)
9. NMR ExperimentsTypes of experiments and
nomenclature
HNCA HN(CO)CA
HN(CA)CO
HNCO
HNCACB HN(CO)CACB
BACKBONE EXPERIMENTS
9. NMR Experiments (cont.)Types of experiments and
nomenclatureSIDE-CHAIN EXPERIMENTS
H(CC)(CO)NHH(CC)NH
HCCH-COSY
HCCH-TOCSY
(H)CC(CO)NH(H)CCNH
10. Putting things together …
Hence: A 3D HCCH-TOCSYlooks like a3D 13C-edited NOESYwithout NOE Cross-peaks.
11. NMR Pulse-SequencesHow 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. SummaryNMR 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 aCOMPLETE 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 of3D EXPERIMENTS employing tailored NMR PULSE
SEQUENCES.NMR pulse-sequences are made up of specifically
arrangedBUILDING BLOCKS .