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 R - G - S - T - L - G - S LT G S S R G

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

COSY: One coupling H N—C H A B C H H H H

R-COSY: Add A 2nd Coupling H N—C—C H A B C H H H N—C—CH 3 H H H

DR-COSY: Add A 3rd Coupling H N—C—C H A B C H H H N—C—CH 3 H H H HH

TOCSY: All Coupled Spins H N—C—C—C—COOH HHH HH H N—C—C—C—C—C—NH 3 HHHHH H HHH H N—C—CH 3 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 NOESY Many Types of NOEs ABCDZ Intraresidue Sequential Medium-range (helices) Long Range Use only these to make sequential assignments

Extended Homonuclear 1 H Strategy Same basic idea as 1 H strategy: based on backbone NH Concept: when backbone 1 H overlaps  disperse with backbone 15 N 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 with different side chains Add a 3rd dimension separating out H N overlaps by their 15 N frequency

15 N Dispersed 1 H- 1 H TOCSY 3 overlapped NH resonancesSame NH, different 15 N F1 F2 F3 1 H 1 H 15 N t1t1 t2t2 t3t3 TOCSY HSQC

Heteronuclear ( 1 H, 13 C, 15 N) Strategy One bond at a time - all atoms (except O) Even handles backbone 15 N 1 H overlaps  disperse with backbone C’C  H  C  H  … Het. 3D/4D increases signal resolution 1 H  13 C  15 N  1 H 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 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 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

1 H- 1 H Distances From NOEs ABCDZ Intraresidue Sequential Medium-range (helices) Long-range (tertiary structure) Challenge is to assign all peaks in NOESY spectra

Approaches to Identifying NOEs 15 N- or 13 C-dispersed 1 H- 1 H NOESY 3D 1H1H 13 C 1H1H 1H1H 15 N 1H1H 1H1H 1H1H 13 C 1H1H 1H1H 1H1H 15 N 1H1H 4D 1H1H 1H1H 2D 1H1H 1H1H 1H1H 3D 1 H- 1 H NOESY

Special NOESY Experiments Filtered, edited NOE: based on selection of NOEs from two molecules with unique labeling patterns. 1H1H 1H1H 13 C Unlabeled peptide Labeled protein Only NOEs at the interface Only NOEs from bound state H H H H k on k off 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 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=OH-N

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 Residual Dipolar Couplings (RDCs)  Requires medium to partially align molecules  Must accommodate multiple solutions  multiple orientations 1H1H 15 N 1H1H HoHo F1 F2 F3 1H1H 13 C 1H1H 1H1H Reports angle of inter- nuclear vector relative to magnetic field H o

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 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 Read the book chapter!