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