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 .