nmr lecture 2

7/31/2019 Nmr Lecture 2

1/15

ProteinNMRPart II


2/15

1. Protein Structures by NMR

NMR, UNLIKE Xray crystallograp

and EM, DOES NOT experimenta

produce a protein structure.

NMR yields Distance Restraints,

which are used to CALCULATE

protein structures.

NMR protein structures are

always ENSEMBLE AVERAGES.

NMR structure calculations yield

MULTIPLE solutions (ensembles)


3/15

2. NMR Structure CalculationH3 Lys10 H2 Arg98 > Int. 0.75

H1 Ser57 H2 Glu42 > Int. 0.45

H2 Asp11 H2 Arg98 > Int. 0.55

H3 Lys72 H2 Arg98 > Int. 0.95

Long-Range/Short-Range

Distance Restraints

Short-Range > 2nd. Structure

Long-Range > Tertiary Structure

(Protein Fold)

Energy Minimization

Assessment of Struct

ural Quality

r.m.s.d., Ramachandran Plot etc.


4/15

3. The Nuclear Overhauser Effect

(NOE Experiments)

Proton-Proton Distances


5/15

4. Problems with Proteins

Spectral Editing (2D or 3D)1

H

1

H

1

H

1

H15N

Selective Labeling

Spectral Overlap


6/15

5. Protein NOEs-sheet NOEs-helical NOEs


7/15

6. yet, how do we know which

one is which?

Goal: Identify the Resonance

Frequencies ofALL Proton,

Carbon and Nitrogen nucleiin a protein.

Backbone Assignment Strategies

Side-Chain Assignment Strategies


8/15

7. Magnetization Transfer

Magnetization transferthrough space > NOE

Magnetization transferthrough bonds > J-coupling

i

i (-1)

1H/15N Correlation (2D)HSQC or HMQC-

type

15N

1H

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


9/15

8. Assignment

ii (-1)

C i

C i (-1)

C i

C i (-1)

HNCACB Experiment(as an example)

What have we learned?

TheAmide Proton resonance frequency

TheAmide Nitrogen resonance frequency

TheAlphaCarbon resonance frequency

The BetaCarbon resonance frequency

i

i (-1)


10/15

9. NMR ExperimentsTypes of experiments and nomenclature

HNC

A

HN(CO)CA

HN(CA)C

O

HNCO

HNCACB HN(CO)CACB

BACKBONE EXPERIMENTS


11/15

9. NMR Experiments (cont.)Types of experiments and nomenclature

SIDE-CHAIN EXPERIMENTS

H(CC)(CO)NHH(CC)N

H

HCCH-COSY

HCCH-TOCSY

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

H


12/15

10. Putting things together

Hence: A 3D HCCH-TOCSYlooks like a

3D 13C-edited NOESY

without NOE Cross-peaks.


13/15

11. NMR Pulse-SequencesHow do we READ them?

What do they MEAN and how do they

WORK?


14/15

12. Lets do one together Can you point outthe basic BUILDING BLOCKS?

Can you determine the MAGNETIZATION TRANSFERPATH?

Can you identify the NMR EXPERIMENT?


15/15

13. SummaryNMR structures are COMPUTED and not experimentally

determined.To calculate an NMR structure we need DISTANCE RESTRAINT

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 of

3D EXPERIMENTS employing tailored NMR PULSE SEQUENCE

NMR pulse-sequences are made up of specifically arranged

BUILDING BLOCKS .

nmr lecture 2

Documents