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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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Presentation Transcript
slide1

Protein

NMR

Part II

slide2

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.

slide3

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.

slide4

3. The Nuclear Overhauser Effect (NOE Experiments)

Proton-Proton Distances

slide5

4. Problems with Proteins

1H

1H

1H

1H

15N

Spectral Overlap

Spectral Editing (2D or 3D)

Selective Labeling

slide6

5. Protein NOEs

-sheet NOEs

-helical NOEs

slide7

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

slide8

7. Magnetization Transfer

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)

slide9

8. Assignment

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

slide10

9. NMR Experiments

Types of experiments and nomenclature

BACKBONE EXPERIMENTS

HNCA

HN(CO)CA

HN(CA)CO

HNCO

HNCACB

HN(CO)CACB

slide11

9. NMR Experiments (cont.)

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

slide12

10. Putting things together …

Hence: A 3D HCCH-TOCSY

looks like a

3D 13C-edited NOESY

without NOE Cross-peaks.

slide13

11. NMR Pulse-Sequences

How do we READ them?

What do they MEAN and how do they WORK?

slide14

12. Let’s do one together …

Can you point outthe basic BUILDING BLOCKS?

Can you determine the MAGNETIZATION TRANSFER PATH?

Can you identify the NMR EXPERIMENT?

slide15

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 .

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