Biomolecular Nuclear Magnetic Resonance Spectroscopy

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

T L R S G G S Basic Strategy to Assign 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 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

A B C D • • • • Z Step 2: Fit Residues In SequenceMinor Flaw: All NOEs Mixed Together Use only these to make sequential assignments Long Range Sequential Intraresidue Medium-range (helices)

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

3 overlapped NH resonances Same NH, different 15N F3 F2 F1 TOCSY HSQC 1H 1H 15N t1 t2 t3 15N Dispersed 1H-1H TOCSY

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

A B C D • • • • Z 1H-1H Distances From NOEs Long-range (tertiary structure) Sequential Intraresidue 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

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

1H 1H Labeled protein Unlabeled peptide 13C 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 kon H koff H H Only NOEs from bound state Identifying Unique NOEs • Filtered, edited NOE:based on selection of NOEs from two molecules with unique labeling patterns.

C=O H-N Hydrogen Bonds • 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 FromScalar Couplings • Must accommodate multiple solutions multiple J values • But database shows few occupy higher energy conformations

F3 F2 F1 Orientational Constraints From Dipolar (D) Couplings Ho Reports angle of inter-nuclear vector relative to magnetic field Ho 1H 1H 13C 15N 1H 1H 15N 1H • 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 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

Biomolecular Nuclear Magnetic Resonance Spectroscopy