Introduction to biological NMR Dominique Marion Institut de Biologie Structurale Grenoble France.
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Transcript of Introduction to biological NMR Dominique Marion Institut de Biologie Structurale Grenoble France.
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
€
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