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 S R G 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 1 H 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
•
2 nd dimension resolves overlaps, 3D rare 1 H 1 H 1 H
Step 1: Identify Spin System
Step 2: Fit Residues In Sequence
Minor Flaw: All NOEs Mixed Together
Use only these to make sequential assignments Sequential
Long Range Intraresidue
A B C D
• • • •
Z
Medium-range (helices)
Extended Homonuclear 1 H Strategy
•
Same basic idea as 1 H strategy: based on backbone NH
•
Concept: when backbone 1 H disperse with backbone 15 N overlaps
•
Use Het. 3D to increase signal resolution 1 H 1 H 15 N
15 N Dispersed 1 H 1 H TOCSY 3 overlapped NH resonances Same NH, different 15 N F2 F1 F3 TOCSY HSQC 1 H 1 H 15 N t 1 t 2 t 3
Heteronuclear ( 1 H, 13 C, 15 N) Strategy
•
Assign resonances for all atoms (except O)
•
Even handles backbone 15 N 1 H overlaps disperse with backbone C’C
a
H
a
C
b
H
b
…
•
Het. 3D/4D increases signal resolution 1 H 13 C 15 N 1 H
•
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 15 N, 13 C, [ 2 H] enrichment
High expression in minimal media (E. coli)
Extra $ ($150/g 13 C-glucose, $20/g 15 NH 4 Cl
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
1 H 1 H Distances From NOEs Sequential Long-range (tertiary structure) 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
•
1 H 1 H NOESY
•
15 N- or 13 C-dispersed 1 H 1 H NOESY 3D 2D 3D 1 H 1 H 1 H 1 H 1 H 4D
Identifying Unique NOEs
•
Filtered, edited NOE:
based on selection of NOEs from two molecules with unique labeling patterns.
Unlabeled peptide Labeled protein Only NOEs at the interface
•
Transferred NOE:
based on: 1) faster build-up of NOEs in large versus small molecules; 2) signal of free state when in excess and exchanging quickly
H H k on k off H H 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
6 Hz Dihedral Angles From Scalar Couplings • • • •
Must accommodate multiple solutions
multiple J values
But database shows few occupy higher energy conformations
Orientational Constraints From Dipolar (D) Couplings H o Reports angle of inter nuclear vector relative to magnetic field H o F2 F1 F3
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