Transcript Center for Structural Biology

01/20/03
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
FROM ASSIGNMENT TO STRUCTURE
Sequential resonance assignment
strategies
NMR data for structure determination
Structure calculations
Properties of NMR structures
Basic Strategy to Assign
Resonances in a Protein
1. Identify resonances for each amino
acid
T G L S
R G
S
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
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
Step 2: Fit Residues In Sequence
Minor Flaw: All NOEs Mixed Together
Use only these to make
sequential assignments
Long Range
Sequential
Intraresidue
A
B
C
Medium-range
(helices)
D
••••
Z
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 resonances
Same NH, different 15N
F2
TOCSY HSQC
1H
1H
t1
t2
15N
t3
F1
F3
Heteronuclear (1H,13C,15N) Strategy
• Assign resonances for all atoms (except O)
• Even handles backbone 15N1H overlaps
disperse with backbone C’CaHaCbHb…
• Het. 3D/4D increases signal resolution
1H
13C
15N
1H
• Works on bigger proteins because 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
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 Overview
NMR Experimental Observables
Providing Structural Information
• Backbone conformation from chemical
shifts (Chemical Shift Index- CSI)
• Distance constraints from NOEs
• Hydrogen bond constraints
• Backbone and side chain dihedral angle
constraints from scalar couplings
• Orientation constraints from residual
dipolar couplings
1H-1H
Distances From NOEs
Long-range
(tertiary structure)
Sequential
Intraresidue
A
B
C
D
••••
Z
Medium-range
(helices)
Challenge is to assign all peaks in NOESY spectra
Protein Fold Without Full
Structure Calculations
1. Determine secondary structure
•CSI directly from assignments
•Medium-range NOEs
2. Add key long-range NOEs to fold
Approaches to Identifying NOEs
• 1H-1H NOESY
2D
3D
•
15N-
or 13C-dispersed
1H-1H NOESY
3D
4D
1H
1H
1H
1H
1H
Identifying Unique NOEs
• Filtered, edited NOE:
Labeled
protein
based on selection of NOEs Unlabeled
peptide
from two molecules with
unique labeling patterns.
Only NOEs at the interface
• Transferred NOE:
H
H
based on: 1) faster build-up
H
kon
of NOEs in large versus
small molecules; 2) signal
koff
H
of free state when in excess
and exchanging quickly
Only NOEs from bound state
Hydrogen Bonds
C=O
H-N
• NH chemical shift to low field
• 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
Dihedral Angles From
Scalar Couplings
•
•
• •
6 Hz
 Must accommodate multiple solutions multiple J values
But database shows few occupy higher energy conformations
Orientational Constraints From
Dipolar (D) Couplings
Ho
Reports angle of internuclear vector relative
to magnetic field Ho
F2
F3
F1
 Must accommodate multiple solutions multiple orientations
NMR Structure Calculations
• Objective is to determine all conformations
consistent with the experimental data
• Programs that only do conformational search
may lead to bad geometry  use simulations
guided by experimental data
 Force fields knocked out of balance
 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 NOEs =
low values of RMSD
Large # of NOEs for
key hydrophobic side
chains
Assessing the Quality
of 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