2. Proteins Have Too Many Signals!
~500 resonances
1H NMR Spectrum of Ubiquitin

3. Regions of the 1H NMR Spectrum

4. Resolve Peaks By Multi-D NMR
A BONUSregions in 2D spectra provide protein fingerprints
If 2D cross peaks overlap go to 3D

5. Challenges For Determining Protein Structures Using NMR
Proteins have thousands of signals
Assign the specific signal for each atom
Thousands of interactions between atoms- also need to be assigned
Need to transform representation from spectrum through interactions between atoms to spatial coordinates

6. Solutions to the Challenges
Increase dimensionality of spectra to better resolve signals: 123 4
Detect signals from heteronuclei (13C,15N)
Better resolve signals- different overlaps
More information to identify signals

7. The Strategy to Assign All of the Resonances in a Protein
Identify resonances for each amino acid
Put amino acids in order
- Sequential assignment (R-G-S,T-L-G-S)
- Sequence-specific assignment
R - G - S - T - L - G - S

8. Homonuclear Assignment Strategy
1H strategy: based on backbone NH (unique region of spectrum, greatest dispersion of resonances, least overlap)
Scalar coupling to identify resonances, dipolar couplings to place in sequence
Concept: build out from the backbone to determine the side chain resonances
2nd dimension resolves overlaps, 3D rare
1H H H

9. Step 1: Identify Spin System

10. Step 2: Fit Residues In Sequence Minor Flaw: All NOEs Mixed Together
Use only these to make sequential assignments
Tertiary Structure
Sequential
Intraresidue A B C D Z
• • • •
Medium-range
(helices)

11. Double-Resonance Experiments Increases Resolution/Information Content
15N-1H HSQC R R
-15N - Ca- CO -15N - Ca H H

12. Extended Homonuclear 1H Strategy
Same basic idea as 1H strategy- based on backbone NH
Concept: when backbone 1H overlaps  disperse with backbone 15N
3rd dimension increases signal resolution
1H H N

13. 3 overlapped NH resonances
15N Dispersed 1H-1H TOCSY
3 overlapped NH resonances
Same NH, different 15N F1 F2 F3
1H 1H 15N t1 t2 t3
TOCSY HSQC

14. 1H-1H TOCSY

16. Heteronuclear (1H,13C,15N) Strategy
Strategy based on backbone 15N1H (13CO)
Concept: when backbone 15N1H overlaps disperse with backbone C’CaHaCbHb…
3rd/4th dimension increase signal resolution
1H C N H
3D/4D double/triple resonance NMR

18. Heteronuclear Assignments: Backbone Experiments
Names of scalar experiments based on atoms detected

19. Heteronuclear Assignments: Side Chain Experiments

32. Figure 20: Example of triple resonance spectra, the HNCO/HN(CA)CO combination of ubiquitin
Figure 21: Sequential connections using HNCACB & HN(CO)CACB on ubiquitin

34. Key Points about Heteronuclear (1H,13C,15N) Strategy
Most efficient, but expts. more complex
Enables study of large proteins (TROSY)
Bonus: sequential assignments
Requires 15N, 13C, [2H] enrichment
High expression in minimal media (E. coli)
Extra $$ (~$200/g 13C6-glucose)

35. Practical Issues: Hardware
Magnet: homogeneous, high field
Electronics: stable, tunable
Environment: temperature, pressure, humidity
Environmental: stray fields
Visit to the Biomolecular NMR Center? Today at 3 PM/ SB party at 4!

36. Practical Issues: Sample Preparation
1D 50 μM
Concentration: Cryoprobe!
nD 400 μM
Volume: 200 μl, 500 μl, 2 mls
Quantity: @ 20kDa  1 mM = 10 mgs
Purity > 95%, buffers
Sensitivity () isotope enrichment (15N, 13C)

37. Practical Issues: Solution Conditions
Variables: buffer, ionic strength, pH, T
Binding studies: co-factors, ligands
H20/D20 exchange- requires unfolding
NO CRYSTALS!

38. Practical Issues: Molecular Weight
30-40 kD for 3D structure Domains
>100 kD: uniform deuteration, residue- and site-specific, atom-specific labeling
Symmetry reduces complexity
2 x 10 kD ≠ 20 kD
TROSY- an experimental approach to higher sensitivity for large systems

39. Large Scalar Couplings  Less Sensitive to MW of the Protein
Superior to 1H homonuclear strategy: H-H couplings <20 Hz
Mixing is faster so less time for signal to relax