Introduction to biological NMR

Dominique MarionInstitut de Biologie Structurale

Grenoble France

Presentation outline

Structural investigation by NMR

NMR spectral parameters

The NMR spectrometer

Two dimensional NMR

Protein HSQC

NMR resonance assignment

NMR structure calculation

Protein-ligand interaction

Molecular motion and relaxation

Structural investigations by NMR

(1) Sample preparation

(a) Optimization of the bacterial expression

(b) Optimization of the protein expression

(c) Labelling [15N] or [15N-13C] or [15N-13C-2H]

(2) NMR experiment recording

(a) Preliminary 2D experiments to optimize experimental conditions

(b) 2D homonuclear experiments (< 80 aa) or 3D triple resonance experiments (>80 aa)

(3) Sequential resonance assignment

(a) Backbone resonances

(b) Side-chain resonances

Structural investigations by NMR

(4) Collection of structural restraints

(a) Internuclear distances (nOe)

(b) Dihedral angles (J-coupling)

(c) Internuclear vector orientations (RDC)

(5) Structure calculation and refinement

(a) Simulated annealing

(b) Structure refinement (MD simulation)

(c) Structure validation (NMR statistics)

(6) Complementary studies

(a) Protein dynamics (Relaxation and echange)

(b) Interaction with partners (ligands…)

NMR spectral parameters

nOe

RDC

Line-width

Shielding Chemical shift

Scalar interaction J-coupling

Nuclear Overhauser effectDipolar interaction

Residual dipolar coupling

Relaxation

NMR spectral parameters

Line-width

J (Hz)

J-coupling

J+D (Hz)

RDC

nOe

Nuclear Overhauser effect

0

(ppm)

Chemical shift

Chemical shift: ring current

Upfield shifted resonance

Downfield shifted resonance

J-coupling (scalar coupling)

J (Hz)

A X

J (Hz)

Nucleus

Electronic cloud

Nuclear spin

Electronic spin

H —— C

1JCH > 0

J-couplings in 15N13C labelled proteins

1JC’N=15Hz

1JNC=11Hz

1JCC=35Hz

1JCC’=55Hz

1JNH=92Hz

1JCH=140Hz

2JNC=7Hz

2JNC’ < 1Hz

Nuclear Overhauser effect

Relaxation in NMR: processes that allow the magnetization to return to equilibrium

Origin: modulation of a spin interaction by the molecular motion

Relaxation mechanisms in NMR: Dipole-dipole interaction Chemical shift anisotropy

nOe (Nuclear Overhauser effect) Transfer of nuclear magnetization from I to S via dipolar cross-relaxation

I

S

rIS

B0

€

DIS = k1

rIS3 3cos2θ IS −1( )

Nuclear Overhauser effect

Energy diagram for a two-spin system.

The levels are populated according to a Boltzman distribution law.

A radiofrequency field saturatesthe A transitions: The corresponding populations are equalized.

In small molecules with a fast tumbling rate, the transition at high frequency W2 is efficient. A population increase is observed for spin A.

In large molecules with a slow tumbling Rate, the transition at low frequency W0 is efficient. A population decrease is observed for spin A.

I

S

rIS

B0

€

DIS = k1

rIS3 3cos2θ IS −1( )

The sign and strength of the dipolar coupling interaction between I and S depends on the relative orientation of thenuclei with respect to B0.

Residual dipolar coupling

Isotropic solution Weakly aligned medium

All orientation of the IS vector are equally likely.The dipolar coupling averages to zero.No structural information

The proteins become weakly aligned.The dipolar coupling does not average exactly to zero.RDC structural information

Residual dipolar coupling

Alignment tensorDescribes the preferential orientation of the protein

Measured RDCsDepends upon the orientation of the internuclear vector with

respect to the alignment tensor.

B0

J-coupling vs RDC

J-couplings provide informationon the relative orientation of the

two internuclear vectors

RDCs provide informationon the absolute orientation ofeach internuclear vector with

respect to a common molecularreference frame

Experimental measurement of J-coupling and nOe

Signal presaturationbefore spectrum recording

Continuous irradiationduring spectrum recording

NMR spectrometer

Superconducting magnet

NMR consoleRf generation

and amplification

WorkstationSpectrometer

control

NMR probe

Superconducting magnet

Dewar Insulation

Liquid nitrogen

Liquid helium

Main magnet coil

Magnet legs

NMR detection probe

Sample

Two-dimensional NMR [1]

Jean Jeener, AMPERE Summer School in Basko Polje, Yugoslavia, September 1971

Preparation MixingEvolution Detection

t1 t2

The preparation and the mixing perioddo not change during the experiment.

Two-dimensional NMR [2]

Preparation Mixing

Evolution

Detection

t2

t1

t2

t1

t2

t1

t2

The receiver is open during the detection

but not during the evolution

Two-dimensional NMR [3]

t1

t2Along t2, all the data pointsare recorded in real time.

Along t1, each data pointrequires a new experiment.

t1

t2

Two-dimensional NMR [4]

Strong signal at the beginning

Weak signalat the end

(thermal noise)

Two-dimensional NMR [5]

Fourier transform along the rows Fourier transform along the columns

t1

t2 t2

t1

Two-dimensional NMR [5]

t2

t1

F2

F1

F2

t1

Two-dimensional NMR [6]

Chemical reaction

A + X B + Y

Reactant

Step 1: identification of the reactants

Product

Step 3: identification of the products

Step 2: chemical reaction

A BMore frequently:equilibrium reaction

Two-dimensional NMR [7]

Correlation spectroscopy

Reactant Product

Preparation MixingEvolution Detection

t1 t2

Step 0: preparation of the reactants

0

Step 1: identification of the reactants

1

Step 2: chemical reaction

2

Step 3: identification of the products

3

Two-dimensional NMR [8]Correlation spectroscopy

A B

Preparation MixingEvolution Detection

t1 t2

F1

F2

A

B

ABA BA AB AB B

Diagonal peaks

Cross-peaks

Two-dimensional NMR [9]

€

S t1, t2( ) = A⋅ exp iΩt( )⋅ exp −R2t( )

1D NMR signal

(in the absence of relaxation)

€

S t1, t2( ) = A⋅ cos Ωt( ) + isin Ωt( )( )

The NMR signal is always described as a complex number€

S t1, t2( ) = A⋅ exp iΩ1t1( )

€

S t1, t2( ) = A⋅ exp jΩ1t1( )⋅ exp iΩ2t2( )

2D NMR signal

cossin

xy

z

Two-dimensional NMR [10]

€

S t1, t2( ) = A⋅ exp jΩ1t1( )⋅ exp iΩ2t2( )

Amplitude modulation

2D NMR signal

Cos (1t1) Cos (2t2) Cos (1t1) Sin (2t2)

Sin (1t1) Sin (2t2)Sin (1t1) Cos (2t2)

RR RI

IR II

Hypercomplex data

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S t1, t2( ) = A⋅ exp i Ω1t1 + Ω2t2( )( )

Two-dimensional NMR [11]

2D NMR signal

Cos (1t1) Cos (2t2) Cos (1t1) Sin (2t2)

Sin (1t1) Sin (2t2)Sin (1t1) Cos (2t2)

RR RI

IR II

Quadrature detection (States Method)

Preparation MixingEvolution Detection

t1 t2

Prep +xPrep +y

1H-15N correlation spectrum of a protein

1D cross-section along the 1H dimension

1D cross-section along the 15N dimension

1H-15N correlation spectrum of a protein

Folded protein

175 residue imipenem-acylated L,D-transpeptidase from B. subtilisLecoq et al

Structure 20, 850-861 (2012).

Disordered protein

179 residue fragment of hepatitis C virus non-structural protein 5A Feuerstein et al Biomol. NMR Assign. 5, 241-243 (2011).

Glycine residues

NMR resonance assignmentGoal: Connecting

a nucleus in the protein

a resonance in the spectrum

The useful information is not the absolute position of a piece…. But the connectivity with its neighbors.

The jigsaw puzzle analogy for NMR resonance assignment

Two pieces have been already successfully matched

Their shape fits roughly the profile of the already matched pair

But only one piece could be anchored effortlessly

This strategy is repeated for all future candidates.

When the puzzle is nearly complete, the location of the remaining pieces can be easily deduced…

Two pieces are possible candidates as neighbors on the right hand side

NMR resonance assignment

NMR resonance assignment

Once the resonance assignement has been obtained, the location of the secondary structure elements (-helices and -sheets) can be determined… without computing the complete NMR structure.

Protein secondary structure prediction

TALOS + : Empirical prediction of protein [ ] backbone torsion angles using HN, HA, CA, CB, CO, N chemical shift assignments

Secondary structure elements in the computed structure

-helices

NMR structure calculations [1]

Collecting conformational restraints

Distance restraints

nOe between nearby hydrogens

Possible pitfalls and difficulties:– multi-spin effect or spin diffusion– conformational averaging (missing nOe)– required distance calibration

Long-range and small nOe carry more structural information

Separation into 3 different classes:– strong nOe (< 2.8 Å)– medium nOe ( < 3.4Å)– small nOe

NMR structure calculations [2]

Collecting conformational restraints

Dihedral angles

Vicinal 3J coupling constantKarplus relationship

Chemical shifts allow the identification of secondary structure elements

Chemical shift index (CSI method) /Talos

Finding a suitable alignment mediumProtein solubility / possible alteration of the conformation

Residual dipolar coupling

NMR structure calculations [3]

Traditional approach for structure calculation

(a)Collecting assigned structural information

(b) Start from a random conformation

(c) Restrained molecular dynamic with

a simplified force field.

(d) Refinement of the structure with

a complete force field and water molecules.

Automated methods for structure calculation

Automated NOESY assignment during structure calculation

NMR structure calculations [4]

Automated methods for structure calculation

NMR structure calculations [5]

Disordered N- and C-terminiDisordered loop

Bacillus subtilis l,D-Transpeptidase169 amino-acids

Ribbon representation

-sheets

-helices

NMR and Refinement Statistics for NMR Structures

Total NOE 3,191

Intraresidue 1,479

Interresidue 1,712

Sequential (|i – j| = 1) 681

Medium-range (|i – j | < 4) 325

Long-range (|i – j| > 5) 706

Total dihedral angle restraints 286

143

143

Total RDC 169

NH 85

CH 84

Qualitative RDC agreement (%) 17

Lecoq L et al. 2012. Dynamics Induced by -Lactam Antibiotics in the Active Site of Bacillus subtilis l,D-Transpeptidase. Structure/Folding and Design 20: 850–61.

Bacillus subtilis l,D-Transpeptidase169 amino-acids

Bacillus subtilis l,D-Transpeptidase169 amino-acids

Violations (mean and SD)

Distance constraints (Å) 0.062 ± 0.005

Dihedral angle constraints (º) 1.87 ± 0.03

Max. dihedral angle violation 17

Max. distance constraint violation 1.61

Deviations from idealized geometry

Bond lengths (Å) 0.0068

Bond angles (º) 0.97

Impropers (º) 2.34

Average pairwise rmsd (Å)

Heavy 0.70 ± 0.10

Backbone 0.39 ± 0.09

Is the calculated structurein agreement with the

experimental data?

Is the covalent geometryof the polypeptidic chain

not distorted?

What is the scattering withinthe set of structures that have

been calculated?

NMR and Refinement Statistics for NMR Structures

Lecoq L et al. 2012. Dynamics Induced by -Lactam Antibiotics in the Active Site of Bacillus subtilis l,D-Transpeptidase. Structure/Folding and Design 20: 850–61.

NMR vs X-rays

Protein-ligand interaction

Addition of the ligand to the protein sampleObservation of the protein spectrum

1D NMR or fast 2D NMR

P P

PL

PL

P P

PL

PL

P + L PL

P + L PL

Slow exchange

Tight binding

Fast exchange

Weak binding

Protein-ligand interaction

The ligand is added to the protein:Some chemical shift variations are observed on the protein.They are located primarilyat the binding interface

A paramagnetic tag is attached to the ligandLine-broadenings are observed on the proteinat the binding interface.

Nuclear Overhauser effect can be observedbetween nuclei in the protein and in the ligand.Discrimination of intra- and intermolecular nOeis possible by means of isotopic labelling.

Residual dipolar couplings can be measured forThe two partners and the complex. If differencesare observed, they can be explained by changesin the preferential orientation of the 2 molecules

Protein dynamics by NMR

Protein function important role of the flexibility

Protein dynamics = time dependent-fluctuations over a wide range of time scale.

Ligand binding

Catalytic enzymes

Folding pathways

Aggregation

Thermostability

Molten globuleMisfolding

Conformational entropy

Excited states

NMR observables and protein motions

10-12 10-9 10-6 10-3 1 103

T1, T2, nOe

RDC

CMPG

EXSY

RT NMRRT NMR

Sidechain

rotation

Proteinglobal

tumblingProtein folding

Enzymatic reactions

Ligand binding

Nuclear spin relaxation Relaxation dispersion Real-time NMR

Time(sec)

Inverted population

What is NMR relaxation?

Boltzmann equilibrium

Magnetization recovery

1 – 2 exp(-t/T1)

Longitudinalrelaxation time

Molecular motion and relaxation

Molecular motions in the liquid-state:

Global molecular tumblingInternal fluctuations

(side-chains, domains)

I

S

rIS

B0

€

DIS = k1

rIS3 3cos2θ IS −1( )

Molecular motions modulate the spin interactions

Here the dipolar interactionbetween spin I and S

The fluctuations of the spin interactioncreate a local fluctuating magnetic field.

This fluctuating magnetic fields push themagnetization toward its equilibrium.

Mz=Mz0 and Mx=My=0

Molecular motion and relaxation

C ––– H

c

C –

–– H

i

C –

–– H

S2

NMR relaxation provides information:

On the speed of the molecular rotationOn the speed of internal motionsOn the amplitude of internal motions

LinkerTailsModules

Molecular motion and relaxationO

rder

para

mete

r (S

2)

Protein sequence

1.0

0.8

0.0

Protein made of two domains connected by a small linker

Presentation outline

Structural investigation by NMR

NMR spectral parameters

The NMR spectrometer

Two dimensional NMR

Protein HSQC

NMR resonance assignment

NMR structure calculation

Protein-ligand interaction

Molecular motion and relaxation