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

H3 Lys10 H2 Arg98 > Int. 0.75

H1 Ser57
H2 Asp11 H3 Lys72

H2 Glu42 > Int. 0.45

H2 Arg98 > Int. 0.55 H2 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)
1 1

15N

Selective Labeling

5. Protein NOEs
-helical NOEs -sheet 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)

HNCA Experiment (3D) HNCACB Experiment (3D)

15N

HSQC or HMQCtype

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(CO)CA

HN(CA)C O

HNCO

HNCAC B

HN(CO)CAC B

9. NMR Experiments (cont.)

Types of experiments and nomenclature SIDE-CHAIN EXPERIMENTS

(H)CCN H(CC)N H H

(H)CC(CO)NH H(CC)(CO)NH

HCCHCOSY

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. Lets 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
Distance restraints are provided by NOE EXPERIMENTS.
NOE experiments are SPECTRAL EDITED and recorded in 3D.

NMR structures are COMPUTED and not experimentally determined. To calculate an NMR structure we need DISTANCE RESTRAINTS

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 .