Transcript Slide 1
BCHM 313 – Physical Biochemistry
Dr. Michael Nesheim (Coordinator)
Rm. A210 Botterell
Dr. Steven Smith (Co-coordinator)
Rm. 615 Botterell
Dr. Susan Yates
Rm. 623 Botterell
BCHM 313 – Physical Biochemistry
Topics
1. Protein NMR – Smith 2. Macromolecular Crystallography – Yates 3. Hydrodynamics – Nesheim 4. Equilibrium Binding – Nesheim 5. Enzyme Kinetics – Nesheim 6. Spectroscopy – Nesheim
Evaluation
Midterm test – 35% Final exam – 65%
Protein NMR Spectroscopy
Determining three-dimensional structures and monitoring molecular interactions
(http://pldserver1.biochem.queensu.ca/~rlc/steve/313/)
Outline
• N-dimensional NMR • Resonance assignment in proteins • NMR-based structure determination • Molecular interactions Reference textbooks: - Lehninger - others available in my office.
Nuclear Magnetic Resonance (NMR)
• • MRI – Magnetic Resonance Imaging (water) • In-vivo spectroscopy (metabolites) • Soild-state NMR (large structures)
Solution NMR
• Chemical structure elucidation - Natural product chemistry - Synthetic organic chemistry – analytical tool of choice for chemists • Biomolecular structural studies (3D structures) -
Proteins
- DNA and Protein/DNA complexes - Polysaccharides • Molecular interactions - Ligand binding and screening (Biotech and BioPharma) •
Nobel prizes
(3): Felix Bloch, 1952 (Physics); Richard Ernest, 1991 (Chemistry); Kurt Wuthrich, 2002 (Chemistry)
Spectroscopy & Nuclear Spin
•Absorption (or emission) spectroscopy (IR, UV, vis). Detects the absorption of radiofrequencies (electro-magnetic radiation) by certain nuclei in a molecule - NMR • Unfortunately, some quantum mechanics are needed to understand it • Only nuclei with
spin number
(
I
) 0 can absorb/emit electro magnetic radiation • Nuclei with even mass number & even number of protons ( 12 C):
I
= 0 • With even mass number & odd number of protons ( 14 N):
I
3….
• With odd mass number ( 1 H, 15 N, 13 C, 31 P):
I
= 1/2, 3/2 …….
= 1, 2, • Spin states of nucleus (m) are quantified: m
I
= (2
I
+ 1)
Thus, for biologically relevant nuclei, two spin states exist: 1/2, 1/2
Spin ½ Nuclei Align in Magnetic Fields
B o Energy Efficiency factor nucleus
D E = h g B o /2
Constants Strength of magnet
• In ground state all nuclear spins are disordered – no energy difference
degenerate
• Since nuclei are small magnets, they orient when a strong magnetic field is applied –
small excess aligned with field (lower energy)
Nucleus 1 13 15 31 H C N P
Intrinsic Sensitivity of Nuclei
g
2.7 x 10 6.7 x 10 -2.7 x 10 1.1 x 10 8 7 7 8
%
Natural 99.98
1.11
0.36
100.
Relative Abundance Sensitivity 1.0
0.004
0.0004
0.5
Resonance: Perturb Equilibrium
B o β α
D E
1. equilibrium Efficiency factor nucleus
H 1 h n = D E
2. pump in energy
D E = h g B o /2
Constant Strength of magnet β α 3. non-equilibrium
Return to Equilibrium (Relax)
β α
D E
3. Non-equilibrium
h n = D E
4. release energy (detect) β α 5. equilibrium
Magnetic Resonance Sensitivity
Sensitivity (S) ~
D(
population)
S ~ D N =
N β N α
= e D E/kT
Efficiency factor nucleus
D E = h g B o /2 • D E is small - @ r.t. ~1:10 5 Thus, intrinsically low sensitivity
*Need lots of sample Constant Strength of magnet
Increase sensitivity by increasing magnetic field strength
Energy/Frequency Relationship
For a particular nucleus: D E = h n D E = h g B /2 n = g B /2 Have to consider
precession
since: 1) nucleus has inherent of a large external magnet generates a
torque spin
which results in and 2) application
precession
w
o
m Precession occurs around B termed Larmor frequency ( w ) .
at frequency, n = g B /2 = w
B o
Reason why 14.1 Telsa magnet often called 600 MHz magnet.
B o
a b
Bulk Magnetization
Two spins
z
All spins
x z y z x
M o
y y
Sum
z x x y
y
B
z
Power of Fourier Transform
z x
90 RF pulse
x
t
z x y y
w = g B
A t
f
w NMR frequency Fourier Transform Variation of signal at X axis vs. time
Pulse Fourier Transform NMR
z z z z x
90 RF pulse t
x x y y y y
w 1 w 2 = g B = g B
A
f
w
2
w
1
NMR frequency domain Spectrum of frequencies
t
Fourier Transform NMR time domain Variation in amplitude vs. time
x
Pulse FT NMR Experiment
90º pulse Experiment equilibration equilibration acquisition (t) detection of signals Data Analysis FID Fourier Transform Time domain (t)
NMR terminology
• Magnetic field (B ) felt by each nucleus affected by its local electronic environment - big difference between B (MHz) and B local (hundreds of Hz) ie. parts per million (ppm, ) • Use relative scale and refer all signals in spectrum to a signal from a reference compound (DSS) = w w ref w ref
B o
a b
Summary
All spins
z
}
y x y
Sum
z
M o
x y z
D E = h g B o /2
z x
90 RF
x
t D E = h n
z x y y
n = g B = w
FID – time domain
Summary (cont)
FID – time domain 10 Chemical shift – relative scale frequency domain ppm 0
NMR terminology
Scalar and Dipolar Coupling
Through Space
Coupling of nuclei gives information on structure
Through Bonds
Resonance Assignment
CH 3 -CH 2 -OH OH CH 2
Which signal from which H atoms?
CH 3
The key attribute: Use scalar and dipolar couplings to match the set of signals with the molecular structure
Proteins Have Too Many Signals!
1 H 1D NMR Spectrum of Ubiquitin
~500 resonances 1 H (ppm)
Resolve resonances by multi-dimensional experiments
Examples of Amino Acids
NMR experiment
90º pulse 1D 2D 90º pulse equilibration equilibration acquisition (t) detection of signals
2D detect signals twice (before/after couple)
preparation evolution (t 1 ) mixing acquisition (t 2 )
Same as 1D experiment Transfers between coupled spins
2D NMR: Coupling is the Key
2D detect signals twice (before/after couple)
90º pulse preparation evolution (t 1 ) mixing acquisition (t 2 )
Transfers between coupled spins Same as 1D experiment
t 1 t 2 t 1 t 2
Pulse sequence
2D NMR Spectrum
excitation preparation evolution (t 1 ) mixing acquisition (t 2 )
Either:
Spectrum t 1 t 2
Before mixing Coupled spins
t 1
or:
t 2
After mixing
1D
The Power of 2D NMR
Resolving Overlapping Signals
2 signals overlapped 2D 2 cross peaks resolved
Multi-Dimensional NMR
Built on the 2D Principle
3D detects signals 3 times
90º pulse excitation preparation evolution (t 1 ) mixing evolution (t 2 ) mixing acquisition (t 3 ) t 2 t 1
Same as 1D experiment
t 3
Protein NMR: Practical Issues Hardware:
• Magnet: homogeneous, high field - $$$$ • Electronics: stable, tunable • Environment: temperature, pressure, humidity, stray fields
Sample Preparation:
• Recombinant protein expression (
E. coli
,
Pichia pastoris
etc) •Volume: 300 m L – 600 m L • Concentration: 1D ~ 50 m M, nD ~ 1mM ie. @ 20 kDa, 1mM = 10 mg • Purity: > 95%, buffers • Sensitivity ( g ): isotope enrichment ( 15 N, 13 C)
Protein NMR: Practical Issues (cont.) Solution Conditions:
• Variables: buffer, ionic strength, pH, temperature • Binding studies: co-factors, ligands • No crystals!
Molecular Weight:
• up to 30 – 40 kDa for 3D structure determination • > 100 kDa: uniform deuteration, residue and site-specific, atom specific labeling • Symmetry reduces complexity: 2 x10 kDa 20 kDa
NMR Spectrum to 3D structure?
|
12 1 H (ppm)
|
0
Critical Features of Protein NMR Spectra
• The nuclei are not mutually coupled
Each amino acid gives rise to an independent NMR sub-spectrum, which is much simpler than the complete protein spectrum
• Regions of the spectrum correspond to different parts of the amino acid • Tertiary structure leads to increased dispersion of resonances • chemical shifts associated with each nucleus influenced by local chemical environment – nearby nuclei
Regions of a protein 1 H NMR Spectrum
What would an unfolded protein look like?
Solutions to the Challenges
1. Increase dimensionality of spectra to better resolve signals: 1 2 3 4 2.
Detect signals from heteronuclei ( 13 C, 15 N) Better resolution of signals/chemical shifts not correlated nuclei More information to identify signals Lower sensitivity to MW of protein
1D Protein 1 H NMR Spectrum
Resolve Peaks by Multi-D NMR
A BONUS
regions in 2D spectra provide protein fingerprints If 2D cross peaks overlap
go to 3D
Basic Strategy to Assign Resonances in Protein
1. Assign 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
Acronyms for Basic Experiments
Differ Only in the Nature of Mixing
Homo
nuclear
Hetero
nuclear
Scalar Coupling
(thru-bond)
COSY CO rrelation S pectroscop Y H SQC H eteronuclear TOCSY TO tal C orrelation S pectroscop Y Hetero-TOCSY Dipolar Coupling
(thru-space)
NOESY N uclear O verhauser E ffect ( E nhancement) S pectroscop Y NOESY-HSQC
Homonuclear 1 H Assignment Strategy
• For proteins up to ~ 10 kDa •Scalar couplings to identify resonances/spin systems/amino acids, dipolar couplings to place in sequence • Based on backbone H N (unique region in 1 H spectrum, greatest dispersion of resonances, least overlap) •
Concept:
Build out from the backbone to identify the side-chain resonances (unique spin systems) • 2 nd dimension resolves overlap, 3D rare
Homonuclear 1 H Assignment Strategy
Step 1: Identify Spin System
COSY (3-bond) TOCSY
CH 3 H H H C N – C – C H H Alanine O
a
H H N
Homonuclear 1 H Assignment Strategy
Step 1: Identify Spin System
H 3 C CH 3 C – H H – C – H N – C – C H H Leucine O COSY (3-bond) TOCSY
b
H
b
’H
g
H
’CH 3
CH 3
a
H H N
Homonuclear 1 H Assignment Strategy
Step 1: Identify Spin System
H 3 C CH 3 H C – H H C H H – C – H N – C – C – N – C – C H H O Alanine
open circles
H H Leucine O
closed circles
COSY (3-bond) TOCSY H N H N
a
H
a
H
b
H
b
’H
CH 3
’CH 3
CH 3
g
H
Homonuclear 1 H Assignment Strategy
Step 2: Fit residues in sequence
Minor Flaw: All NOEs mixed together!
Use only these to make sequential assignments Sequential
Long Range Intraresidue
A
•Sequential NOEs H N -H N (i, i + 1) H a -H N (i, i + 1)
B C D
• • • • Medium-range (helices: H a -HN (i, i + 3,4)) )
Z
Homonuclear 1 H Assignment Strategy
Step 2: Fit residues in sequence
H 3 C CH 3 H C – H H C H H – C – H N – C – C – N – C – C H H O H H O NOESY = COSY/ TOCSY +
a
H
a
H
b
H
b
’H
’CH 3
CH 3
b
CH 3
g
H H N H N
Extended Homonuclear 1 H Strategy
• For proteins up to ~ 15 kDa •Same basic idea as 1 H strategy: based on backbone H N •
Concept:
When backbone 1 H overlaps disperse with backbone 15 N • Use heteronuclear 3D experiments to increase signal resolution 1 H 1 H 15 N
Solutions to the Challenges
1. Increase dimensionally of spectra to better resolve signals: 1 2 3 4 2.
Detect signals from heteronuclei ( 13 C, 15 N) Labeling with NMR-observable 13 C, 15 N isotopes Better resolution of signals/chemical shifts not correlated nuclei More information to identify signals Lower sensitivity to MW of protein
Isotopic Labeling
• Require uniform 15 N/ 13 C labeling ie. Every carbon and nitrogen isotopically labeled
How?
• Grow bacteria on minimal media (salts) supplemented with 15 N-NH 4 Cl and 13 C-glucose as soles sources of nitrogen and carbon • lower yields than protein expression than on enriched media, therefore need very good recombinant expression system
Double Resonance Experiments
Increases Resolution/Information Content
Heteronuclear NMR: 15 N-Edited Experiments
Increases Resolution/Information Content
R H 15 N – C
a
– C – 15 N – C
a
H O R
3D Heteronuclear NMR: 15 N-Edited Experiments
+
Extended Homonuclear 1 H Strategy
15 N dispersed 1 H 1 H TOCSY
3 overlapped NH resonances (diagonal) H N (ppm) Same NH, different 15 N TOCSY HSQC 1 H 1 H 15 N t 1 t 2 t 3 F2 F1 F3 R H 15 N – C
a
– C – 15 N – C
a
H O R
Summary of Homonuclear Assignment Strategy
• for proteins up to ~10 kDa (2D homonuclear) and proteins up to ~ 15 kDa ( 15 N-labeling and 3D) • using scalar coupling-type experiments (COSY, TOCSY) assign spin systems/side-chain resonances • Connect amino acids (identified based on spin systems) sequentially using NOE-type experiments and characteristic sequential NOEs (H N -H N (i, i+1); H a -H N (i, i+1))
Heteronuclear ( 1 H, 13 C, 15 N) Strategy
• for larger proteins (backbone assignment: ~70 kDa; full structure determination: ~40 kDa) •Assign resonances (chemical shifts) for all atoms (except O) 15 •Handles overlap in backbone H region disperse with backbone
C’, C
a
, H
a
,C
b
, H
b • Heteronuclear 3D/4D increases resolution
1 H 13 C 1 H 15 N
• Works on bigger proteins because scalar couplings are larger
Heteronuclear ( 1 H, 13 C, 15 N) Strategy
Step 1: Sequence-specific backbone assignment
Assign backbone 1 H, 15 N, C a , C b resonances/chemical shifts and sequentially link amino acids using partner scalar coupling experiments
Step 2: Side-chain assignment
Assign side-chain 13 C & 1 H resonances/chemical shifts using TOCSY-type 3D scalar coupling experiments
** Have complete list of chemical shifts for all 13 C, 15 N, 1 H atoms in protein **
Heteronuclear ( 1 H, 13 C, 15 N) Assignments
Backbone Experiments
Names of scalar experiments based on atoms detected
Consecutive residues!!
NOESY not needed
Heteronuclear ( 1 H, 13 C, 15 N) Assignments
Backbone Experiments
CBCA(CO)NH
-
inter-residue connectivity (HN to previous C
a
, C
b)
HNCACB - intra-residue connectivity (HN to own C
a
, C
b)
Search 15 N planes for 13 C
a
and 13 C
b
chemical shifts 13 C
b
chemical shift R H H – C – H H – C – H H N – C N – C N – C – C H H O H O H H O 13 C
a
chemical shift common 15 N and H N chemical shift in both experiments (found on same 15 N plane)
Heteronuclear ( 1 H, 13 C, 15 N) Assignments
Backbone Experiments
CBCA(CO)NH
-
inter-residue connectivity (HN to previous C
a
, C
b) -
HNCACB intra-residue connectivity and possibly inter-residue (HN to own C
a
, C
b)
Start with unique residue 1. Gly – only C
a
2. Ala – upfield-shifted C
b
(~18 ppm) 3. Thr/Ser – downfield-shifted C
a
& C
b
which are close to each other
Heteronuclear ( 1 H, 13 C, 15 N) Assignments
Side-chain Experiments
Multiple redundancies increase reliability
Heteronuclear ( 1 H, 13 C, 15 N) Assignments
Key Points
• Enables the study/assignment of much larger proteins (up to ~100 kDa) •Scalar coupling-type 3-dimensional experiments only •
Bonus:
Amino acid identification and sequence-specific assignment all at once • Most efficient but experiments are more complex •Requires 13 C, 15 N enrichment (also 2 H) High expression levels on minimal media Increased cost ($150/g 13 C-gluocose; $30/g 15 NH 4 Cl)
Structure Determination Overview
List of chemical shifts for all nuclei in protein ( 1 H, 13 C, 15 N)
NMR Experimental Observables Provide Structural Information
1. Backbone conformation from chemical shifts (Chemical Shift Index – CSI; H a , C a , C b , C’) 2. Hydrogen bond constraints 3. Backbone and side chain dihedral angle constraints from scalar couplings 4. Distant constraints from NOE connectivities
1. Chemical Shift Index
• Comparison of H a , C a , C b , C’ determined chemical shifts from protein to standard random coil chemical shift values • Upfield-shifted H a and C b and downfield-shifted C a and C’ values indicate amino acid residues in an a -helical conformation (requires three consecutive residues displaying this pattern) • Downfield-shifted H a and C b and upfield-shifted C a and C’ values indicate residues in an extended ( b -strand) conformation
2. Hydrogen Bonds
C=O H-N
• Slow rate of exchange of labile H N with solvent •Protein dissolved in 2 H 2 O; H N signals disappear with time •H N groups that are H bonded (i.e. part of secondary structure) will exchange a lot slower than those in loops
6 Hz
3. Dihedral Angles from Scalar Couplings
• • • •
Must accommodate multiple solutions
multiple J values
4. 1 H 1 H Distances from NOEs
Long-range (tertiary structure) Sequential Intraresidue A B C D
• • • •
Z Medium-range (helices)
Challenge is to assign all peaks in NOESY spectra - semi-automated processes for NOE assignment using NOESY data and table of chemical shifts yet still significant amount of human analysis
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
2D 1 H 1 H
•
15
N- or
13
C dispersed
1
H-
1
H NOESY
3D 4D
NMR Structure Calculations Objective:
Determine all conformations consistent with experimental data • Programs that only do conformational search may lead to bad geometry use simulations guided by experimental data • need a reasonable starting structure •Distance restraints arrived at from NOE signal intensities signal is an average of all conformations
NMR Structure Calculations (cont)
1. NOE signals are time & population-averaged (ie. measured on entire sample over period of time) 2. Intensity of NOE signal 1 H 1 H distance (1/r 6 ) NOE distance restraints are given a range of values strong NOE: 0 - 2.8 Å medium NOE: 2.8 – 3.5 Å weak NOE: 3.5 – 5.0 Å
NMR data not perfect: Noise, incomplete data
multiple solutions (conformational ensemble unlike X-ray crystallography with one solution)
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
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
Summary of Protein NMR Structure Determination
Sample preparation with possible isotope labeling
Data collection (scalar coupling and dipolar coupling expts.
Resonance and sequence-specific assignments
Identification and quantification of NOE peaks and intensities and conversion to approx. 1 H 1 H distances
Generation of models consistent with NOE distance constraints, dihedral angle ranges, H-bond distances
Model improvement by inclusion of newly identified NOES using above mentioned models
NMR Structures – Now what?
H31 V19 R25 I34
Monitoring Molecular Interactions
15 N 1 H HSQC G27 A14 Y36 T23 M20 C37 G22 S16 V29 K33 W17 I24 F28 A32 L15 NMR Provides
Site-specific
Multiple probes
In-depth info
Spatial distribution of responses can be mapped on structure Q21 S35 K26 D18 N30
Monitoring Molecular Interactions
Titration followed by 15 N 1 H HSQC
Monitoring Molecular Interactions
Transcription factor (CBP) -oncoprotein (E2A) interaction - collaboration with Dr. David LeBrun (Pathology) Map of chemical shift perturbations on the structure of protein?
Monitoring Molecular Interactions
- Identification of ligand (E2A)-binding site on the structure of the KIX domain of CBP
Monitoring Molecular Interactions
Chemical Perturbation Mapping Structure
Ligand Binding
NMR timescale – 1 sec to 1 x 10 -6 sec 1/k off = t >> 1 sec
slow exchange, superposition of spectra 1/k off = t << 1 x 10 -6 sec
fast exchange, weighted average k on A B k off K diss = [A]/[B] = k off /k on
Ligand Binding
-
Another protein
-
Metal ion
-
Drug or chemical P + L = PL K diss = [P] [L] [PL]
Ligand Binding - exchange
E641, S642, and S670 - Fast exchange (weighted average of free and bound populations) T614 - Intermediate-fast exchange
Ligand Binding
P tot = P + PL L tot = L + PL So……. K diss = [P tot - PL] [L tot [PL] - PL] Plot [L tot ]/[P tot ] vs “
change
” in NMR spectra For fast exchange (weak binding):
obs -
init Change = =
sat -
init [ PL] [P tot ] shifting of resonances in spectra For slow exchange (tight binding): Integral of peak obs Change = = Integral of peak max [ PL] [P tot ] intensity changes in peaks of free and bound forms
Monitoring Molecular Interactions
Binding Constants by NMR
Stronger Weaker Molar ratio of d-CTTCA Fit change in chemical shift to binding equation
Protein Dynamics
Interesting because……..
• Function requires motion/kinetic energy • Entropic contributions to binding events • Protein folding/unfolding • Uncertainty in NMR and crystal structures • Effects on NMR experiments: spin relaxation is dependent on motions know dynamics to predict outcomes and design new experiments
Characterizing Protein Dynamics
Parameters & Timescale
Dynamics from NMR Parameters
• Number of signals per atom:
multiple signals for slow exchange between conformational states
Populations ~ relative stability R ex <
w
(A) -
w
(B) Rate A B
Dynamics from NMR Parameters
• Number of signals per atom:
multiple signals for slow exchange between conformational states
• Linewidths:
narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW)
Linewidths Dependent on Protein MW
A B A B 15 N 15 N 15 N 1 H 1 H
• Same chemical shifts, same structure • Linewidth determined By size of molecule
1 H
• Fragments have narrow linewidths
Dynamics from NMR Parameters
• Number of signals per atom:
multiple signals for slow exchange between conformational states
• Linewidths:
narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW)
• Exchange of H N solvent:
slow timescales (milliseconds to years!)
•
requires local or global unfolding events
•
H N involved in H-bonds exchanges slowly
•
surface or flexible region: HN exchange rapidly
Dynamics from NMR Parameters
• Number of signals per atom:
multiple signals for slow exchange between conformational states
• Linewidths:
narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW)
• Exchange of H N
to years!)
solvent:
slow timescales (milliseconds
• NMR relaxation measurements (ps – ns; m s – ms) •
R 1 (1/T 1 ) spin lattice relaxation rate (z-axis)
• •
R 2 (1/T 2 ) spin spin relaxation rate (xy-plane) Heteronuclear NOE ( 15 N 1 H)
Dynamics to Probe the Origin of Structural Uncertainty
Weak correlation Strong correlation
- Measurements show if high RMSD is due to high flexibility (low S 2 )
NMR and Crystallography NMR
•
Can mimic biological conditions - pH, temp, salt
•
information on dynamics
•
monitor conformational change on ligand binding
•
2
structure derived from limited experimental data
•
need concentrated sample - lots of protein; aggregation issues
•
size limited – ~40kDa for full structure determination
•
more subjective interpretation of data
•
lack of quality factors resolution and R-factor
X-ray
•
Highly automated with more objective interpretation of data
•
Quality indicators (resolution, R)
•
Surface residues and water molecules well defined
•
Huge molecules and assemblies can be determined
•
non-physiological conditions – crystallization difficult
•
need heavy-atom derivatives – production not always trivial
•
snap-shot of protein in time – less indication of mobility
•
flexible proteins difficult to crystallize
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
: Used for weak interactions (ligand in excess) and based on NOEs from bound state passed to free state H H H Only NOEs from bound state k on k off H