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Protein. NMR. Part II. 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

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Protein

NMR

Part II


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.


2. NMR Structure Calculation

Long-Range/Short-Range

Distance Restraints

H3 Lys10

H1 Ser57

H2 Asp11

H3 Lys72

H2 Arg98 > Int. 0.75

H2 Glu42 > Int. 0.45

H2 Arg98 > Int. 0.55

H2 Arg98 > Int. 0.95

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 Experiments)

1H

1H

1H

1H

15N

Spectral Overlap

Spectral Editing (2D or 3D)

Selective Labeling


5. Protein NOEs Experiments)

-sheet NOEs

-helical NOEs


6. … yet, how do we know which one is which? Experiments)

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 Experiments)

i

1H/15N Correlation (2D)

HSQC or HMQC-type

15N

i (-1)

1H

Magnetization transfer through space > NOE

Magnetization transfer through bonds > J-coupling

HNCA Experiment (3D)

HNCACB Experiment (3D)


8. Assignment Experiments)

C i

C i (-1)

C i (-1)

i

C i

i (-1)

HNCACB Experiment

(as an example)

What have we learned?

The Amide Proton resonance frequency

The Amide Nitrogen resonance frequency

The AlphaCarbon resonance frequency

The BetaCarbon resonance frequency

i (-1)

i


9. NMR Experiments Experiments)

Types of experiments and nomenclature

BACKBONE EXPERIMENTS

HNCA

HN(CO)CA

HN(CA)CO

HNCO

HNCACB

HN(CO)CACB


9. NMR Experiments (cont.) Experiments)

Types of experiments and nomenclature

SIDE-CHAIN EXPERIMENTS

(H)CC(CO)NH

(H)CCNH

H(CC)NH

H(CC)(CO)NH

HCCH-COSY

HCCH-TOCSY


10. Putting things together … Experiments)

Hence: A 3D HCCH-TOCSY

looks like a

3D 13C-edited NOESY

without NOE Cross-peaks.


11. NMR Pulse-Sequences Experiments)

How do we READ them?

What do they MEAN and how do they WORK?


12. Let’s do one together … Experiments)

Can you point outthe basic BUILDING BLOCKS?

Can you determine the MAGNETIZATION TRANSFER PATH?

Can you identify the NMR EXPERIMENT?


13. Summary Experiments)

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 .


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