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

Regions of the 1H NMR Spectrum

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

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

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

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 1 2 3 4 5 6 7 R - G - S - T - L - G - S

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 1H 1H

Step 1: Identify Spin System

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)

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

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 1H 15N

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

1H-1H TOCSY

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 13C 15N 1H 3D/4D double/triple resonance NMR

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

Heteronuclear Assignments: Side Chain Experiments

http://students.washington.edu/~baracuda/bioc530/nmr3.html

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

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)

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!

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)

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

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

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