biomolecular nuclear magnetic resonance spectroscopy from assignment to structure sequential...
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Biomolecular Nuclear Magnetic Resonance Spectroscopy
FROM ASSIGNMENT TO STRUCTURE
Sequential resonance assignment strategies
NMR data for structure determination
Structure calculations
Properties of NMR structures
01/26/04
Basic Strategy to Assign Resonances in a Protein
1. Identify resonances for each amino acid
2. 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
LT G S S R G
Homonuclear 1H Assignment Strategy
• Scalar coupling to identify resonances, dipolar couplings to place in sequence
• Based on backbone NH (unique region of spectrum, greatest dispersion of resonances, least overlap)
• Concept: build out from the backbone to identify the side chain resonances
• 2nd dimension resolves overlaps, 3D rare
1H 1H 1H
Step 1: Identify Spin System
COSY: One coupling
H
N—C
H
A
B
C
H
N—C
H
H
N—C
H
R-COSY: Add A 2nd Coupling
H
N—C—C
H
A
B
C
H
N—C—C
H
H
N—C—CH3
H
H
H
DR-COSY: Add A 3rd Coupling
H
N—C—C
H
A
B
C
H
N—C—C
H
H
N—C—CH3
H
H
H
H
H
TOCSY: All Coupled Spins
H
N—C—C—C—COOH
H H H
H H
H
N—C—C—C—C—C—NH3
H H H H H
H H H HH
N—C—CH3
H
A
B
C
Step 2: Fit Residues in Sequence
Peaks in NOESY spectra
Same as scalar coupling peaks
Peaks from residue i to i+1
A - B - C
A B (B C)
A Minor Problem With NOESYMany Types of NOEs
A B C D Z• • • • Intraresidue
Sequential
Medium-range(helices)
Long Range
Use only these to make sequential assignments
Extended Homonuclear 1H Strategy
• Same basic idea as 1H strategy: based on backbone NH
• Concept: when backbone 1H overlaps disperse with backbone 15N
• Use Het. 3D to increase signal resolution
1H 1H 15N
15N Dispersed 1H-1H TOCSY
3 overlapped NH resonanceswith different side chains
Add a 3rd dimension separating out HN overlaps by their 15N frequency
15N Dispersed 1H-1H TOCSY
3 overlapped NH resonances
Same NH, different 15N
F1
F2F3
1H 1H 15Nt1 t2 t3
TOCSY HSQC
Heteronuclear (1H,13C,15N) Strategy
• One bond at a time - all atoms (except O)
• Even handles backbone 15N1H overlaps disperse with backbone
C’CHCH…
• Het. 3D/4D increases signal resolution
1H 13C 15N 1H
• Works on bigger proteins because one bond scalar couplings are larger
Heteronuclear Assignments:Backbone Experiments
Names of scalar experiments based on atoms detected
Consecutive residues!!NOESY not needed
Heteronuclear Assignments:Side Chain Experiments
Multiple redundancies increase reliability
Tutorial on the website
Heteronuclear Strategy: Key Points
• Bonus: amino acid identification and sequential assignments all at once
• Most efficient, but expts. more complex
• Enables study of much larger proteins (TROSY/CRINEPT 1 MDa: e.g. Gro EL)
• Requires 15N, 13C, [2H] enrichment High expression in minimal media (E. coli) Extra $ ($150/g 13C-glucose, $20/g 15NH4Cl)
Structure Determination by NMRStructure Determination by NMR
NMR Experimental Observables Providing Structural Information
• Backbone conformation from chemical shifts (Chemical Shift Index- CSI): ,
• Distance restraints from NOEs
• Hydrogen bond restraints
• Backbone and side chain dihedral angle restraints from scalar couplings
• Orientation restraints from residual dipolar couplings
11H-H-11H Distances From NOEsH Distances From NOEs
A B C D Z• • • • Intraresidue
Sequential
Medium-range(helices)
Long-range(tertiary structure)
Challenge is to assign all peaks in NOESY spectra
Approaches to Identifying NOEsApproaches to Identifying NOEs
• 15N- or 13C-dispersed 1H-1H NOESY 3D 1H
13C
1H1H15N
1H
1H15N
1H
13C
1H13C
1H
13C
1H15N
1H
15N
4D
1H 1H2D
1H 1H 1H3D
• 1H-1H NOESY
Special NOESY ExperimentsSpecial NOESY Experiments
•Filtered, edited NOE: based on selection of NOEs from two molecules with unique labeling patterns.
1H 1H
13CUnlabeled
peptide
Labeledprotein
Only NOEs at the interface
Only NOEs from bound state
H
H
HH
kon
koff
•Transferred NOE: based on 1) faster build-up of NOEs in large versus small molecules; 2) Fast exchange 3) NOEs of bound state detected at resonance frequencies of free state
Backbone Hydrogen BondsBackbone Hydrogen Bonds
• NH chemical shift at low field (high ppm)
• Slow rate of NH exchange with solvent
• Characteristic pattern of NOEs
• (Scalar couplings across the H-bond)
When H-bonding atoms are known can impose a series of distance/angle constraints to enforce standard H-bond geometries
C=O H-N
• • • •6 Hz
Dihedral Angles FromScalar Couplings
Must accommodate multiple solutions multiple J valuesBut database shows few occupy higher energy conformations
Orientational Constraints From Residual Dipolar Couplings (RDCs)
Requires medium to partially align molecules Must accommodate multiple solutions multiple orientations
1H
15N
1H
15N
Ho
F1
F2F3
1H13C
1H
1H
Reports angle of inter-nuclear vector relative to magnetic field Ho
NMR Structure Calculations
• Objective is to determine all conformations consistent with the experimental data
• Programs that only do conformational search lead to bad chemistry use molecular force fields improve molecular properties Some programs try to do both at once Need a reasonable starting structure
• NMR data is not perfect: noise, incomplete data multiple solutions (conformational ensemble)
Variable Resolution of Structures
• Secondary structures well defined, loops variable
• Interiors well defined, surfaces more variable
• Trends the same for backbone and side chains More dynamics at loops/surface Constraints in all directions in the interior
Restraints and Uncertainty
Large # of restraints = low values of RMSD
Large # of restraints for key hydrophobic side chains
Assessing the Qualityof NMR Structures
• Number of experimental constraints
• RMSD of structural ensemble (subjective!)
• Violation of constraints- number, magnitude
• Molecular energies
• Comparison to known structures: PROCHECK
• Back-calculation of experimental parameters
Read the book chapter!
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