Transcript Slide 1

BCHM 313 – Physical Biochemistry
Dr. Michael Nesheim (Coordinator)
[email protected]
Rm. A210 Botterell
Dr. Steven Smith (Co-coordinator)
[email protected]
Rm. 615 Botterell
Dr. Susan Yates
[email protected]
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 (12C):
I=0
• With even mass number & odd number of protons (14N): I = 1, 2,
3….
• With odd mass number (1H, 15N, 13C, 31P): I = 1/2, 3/2 …….
• Spin states of nucleus (m) are quantified: mI = (2I + 1)
Thus, for biologically relevant nuclei, two spin states exist: 1/2, 1/2
Spin ½ Nuclei Align in Magnetic Fields
Bo
Energy
Efficiency factornucleus
DE = h g Bo/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)
Intrinsic Sensitivity of Nuclei
Nucleus
g
% Natural
Abundance
Relative
Sensitivity
1H
2.7 x 108
99.98
13C
6.7 x 107
1.11
0.004
15N
-2.7 x 107
0.36
0.0004
31P
1.1 x 108
100.
1.0
0.5
Resonance: Perturb Equilibrium
β
DE
Bo
1. equilibrium
Efficiency factornucleus
α
H1
hn = DE
DE = h g Bo/2
2. pump in energy
Constant
β
α
3. non-equilibrium
Strength of
magnet
Return to Equilibrium (Relax)
β
DE
3. Non-equilibrium
α
hn = DE
4. release energy (detect)
β
5. equilibrium
α
Magnetic Resonance Sensitivity
Sensitivity (S) ~ D(population)
Nβ
= e-DE/kT
S ~ DN =
Nα
• DE is small - @ r.t. ~1:105
Thus, intrinsically low sensitivity
*Need lots of sample
Efficiency factornucleus
DE = h g Bo/2
Constant
Strength of
magnet
Increase sensitivity by increasing magnetic field strength
Energy/Frequency Relationship
For a particular nucleus:
DE = hn
DE = hgB/2
n = gB/2
Have to consider precession since: 1) nucleus has inherent spin and 2) application
of a large external magnet generates a torque which results in precession
wo
Precession occurs around B at frequency,
termed Larmor frequency (w).
n = gB/2 = w
m
Bo
Reason why 14.1 Telsa magnet often
called 600 MHz magnet.
Bulk Magnetization
Two spins
All spins
Sum
z
x
Bo
b
z
z
y
Mo
x
z
y
a
x
y
y
x
Power of Fourier Transform
z
z
x
90 RF pulse
y
z
t
x
y
x
y
w = gB
B
A
t
f
w
NMR frequency
Fourier
Transform
Variation of signal
at X axis vs. time
Pulse Fourier Transform NMR
z
z
x
x
y
y
z
z
90 RF
pulse
x
t
x
y
y
w1 = gB
w2 = gB
A
f
w2 w1
NMR frequency domain
 Spectrum of frequencies
t
Fourier
Transform
NMR time domain
 Variation in amplitude vs. time
Pulse FT NMR Experiment
90º pulse
Experiment
equilibration
acquisition (t)
equilibration
Data
Analysis
FID
Time domain (t)
detection of signals
Fourier
Transform
NMR terminology
• Magnetic field (B) felt by each nucleus affected by its local electronic
environment - big difference between B (MHz) and Blocal (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 - wref
=
wref
Summary
All spins
Sum
z
b
Bo
}
z
y
z
FID – time domain
x t
y
n = gB = w
DE = hn
z
90 RF
x
y
y
DE = hgBo/2
Mo
x
a
x
z
x
y
Summary (cont)
FID – time domain
frequency domain
10
Chemical shift – relative scale
ppm
0
NMR terminology
Scalar and Dipolar Coupling
Through
Space
Through
Bonds
Coupling of nuclei gives information on structure
Resonance Assignment
CH3-CH2-OH
OH
CH2 CH3
Which signal from which H atoms?
The key attribute: Use scalar and dipolar couplings to
match the set of signals with the molecular structure
Proteins Have Too Many Signals!
1H
1D NMR Spectrum of Ubiquitin
~500 resonances
1H
(ppm)
Resolve resonances by multi-dimensional experiments
Examples of Amino Acids
NMR experiment
90º pulse
1D
equilibration
acquisition (t)
equilibration
2D
90º pulse
preparation
Same as 1D
experiment
detection of signals
2D detect signals twice
(before/after couple)
evolution (t1)
mixing
acquisition (t2)
Transfers between
coupled spins
2D NMR: Coupling is the Key
2D detect signals twice
(before/after couple)
90º pulse
evolution (t1)
preparation
mixing
acquisition (t2)
Transfers between
coupled spins
Same as 1D
experiment
t1
t2
t1
t2
2D NMR Spectrum
excitation
Pulse sequence
preparation
evolution (t1)
mixing
acquisition (t2)
Spectrum
HB
Either:
t1
t2
Before mixing
HA
Coupled spins
or:
t1
After mixing
t2
The Power of 2D NMR
Resolving Overlapping Signals
1D
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 (t1)
Same as 1D
experiment
mixing
evolution (t2)
mixing
t1
acquisition (t3)
t2
t3
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 mL – 600 mL
• Concentration: 1D ~ 50 mM, nD ~ 1mM ie. @ 20 kDa, 1mM = 10 mg
• Purity: > 95%, buffers
• Sensitivity (g): isotope enrichment (15N, 13C)
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, atomspecific labeling
• Symmetry reduces complexity: 2 x10 kDa  20 kDa
NMR Spectrum to 3D structure?
|
12
1H
(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 1H 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 (13C, 15N)
 Better resolution of signals/chemical shifts not
correlated nuclei
 More information to identify signals
 Lower sensitivity to MW of protein
1D Protein 1H 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
L
G
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
Scalar Coupling
(thru-bond)
Homonuclear
Heteronuclear
COSY
HSQC
COrrelation SpectroscopY
Heteronuclear
TOCSY
Hetero-TOCSY
TOtal Correlation SpectroscopY
Dipolar Coupling
(thru-space)
NOESY
Nuclear Overhauser Effect
(Enhancement) SpectroscopY
NOESY-HSQC
Homonuclear 1H 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 HN (unique region in 1H spectrum,
greatest dispersion of resonances, least overlap)
• Concept: Build out from the backbone to identify the
side-chain resonances (unique spin systems)
• 2nd dimension resolves overlap, 3D rare
Homonuclear 1H Assignment Strategy
Step 1: Identify Spin System
COSY (3-bond)
TOCSY
–
– – –
H
H
C
H
N–C–C
=
aH
H H O
Alanine
HN
CH3
Homonuclear 1H Assignment Strategy
Step 1: Identify Spin System
’CH3
COSY (3-bond)
CH3
H3C
CH3
TOCSY
gH
–
– – –
C–H
b’H
bH
H–C–H
N–C–C
H
=
H
aH
O
Leucine
HN
Homonuclear 1H Assignment Strategy
Step 1: Identify Spin System
’CH3
COSY (3-bond)
CH3
H3C
H
H
=
N–C–C
O
–
– – –
H
gH
b’H
H–C–H
bH
–N–C–C
H
H
aH
=
–
– – –
C
CH3
C–H
H
H
CH3
TOCSY
aH
O
Alanine
Leucine
- open circles - closed circles
HN
HN
Homonuclear 1H Assignment Strategy
Step 2: Fit residues in sequence
Minor Flaw: All NOEs mixed together!
Use only these to make
sequential assignments
Long Range
Sequential
Intraresidue
•Sequential NOEs
 HN-HN (i, i + 1)
 Ha-HN (i, i + 1)
A
B
C
D
••••
Medium-range
(helices: Ha-HN (i, i + 3,4)))
Z
Homonuclear 1H Assignment Strategy
Step 2: Fit residues in sequence
’CH3
CH3
H3C
C
H
H
H
=
N–C–C
O
H–C–H
–N–C–C
H
H
bCH3
gH
b’H
bH
aH
=
–
– – –
H
–
– – –
C–H
H
CH3
NOESY =
COSY/TOCSY
+
aH
O
HN
HN
Extended Homonuclear 1H Strategy
• For proteins up to ~ 15 kDa
•Same basic idea as 1H strategy: based on
backbone HN
• Concept: When backbone 1H overlaps 
disperse with backbone 15N
• Use heteronuclear 3D experiments to increase
signal resolution
1H
1H
15N
Solutions to the Challenges
1. Increase dimensionally of spectra to better resolve
signals: 1  2  3  4
2. Detect signals from heteronuclei (13C, 15N)
 Labeling with NMR-observable 13C, 15N 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 15N/13C labeling ie. Every carbon and
nitrogen isotopically labeled
How?
• Grow bacteria on minimal media (salts) supplemented with
15N-NH Cl and 13C-glucose as soles sources of nitrogen and
4
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: 15N-Edited Experiments
Increases Resolution/Information Content
H
R
15N
H
– Ca – C – 15N – Ca
O
R
3D Heteronuclear NMR:
15N-Edited
+
Experiments
Extended Homonuclear 1H Strategy
15N dispersed 1H-1H TOCSY
3 overlapped NH resonances
(diagonal)
HN (ppm)
Same NH, different 15N
F3
F2
F1
TOCSY HSQC
1H
1H
t1
t2
15N
t3
H
R
15N
H
– Ca – C – 15N – Ca
O
R
Summary of Homonuclear Assignment Strategy
• for proteins up to ~10 kDa (2D homonuclear)
and proteins up to ~ 15 kDa (15N-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
(HN-HN (i, i+1); Ha-HN (i, i+1))
Heteronuclear (1H, 13C, 15N) Strategy
• for larger proteins (backbone assignment: ~70 kDa; full
structure determination: ~40 kDa)
•Assign resonances (chemical shifts) for all atoms
(except O)
15N
•Handles overlap in backbone H region
disperse with backbone C’, Ca, Ha ,Cb, Hb
• Heteronuclear 3D/4D increases resolution
1H
13C
1H
15N
• Works on bigger proteins because scalar couplings are
larger
Heteronuclear (1H, 13C, 15N) Strategy
Step 1: Sequence-specific backbone assignment
Assign backbone 1H, 15N, Ca, Cb resonances/chemical
shifts and sequentially link amino acids using partner
scalar coupling experiments
Step 2: Side-chain assignment
Assign side-chain 13C & 1H resonances/chemical shifts
using TOCSY-type 3D scalar coupling experiments
** Have complete list of chemical shifts for all 13C,
15N, 1H atoms in protein **
Heteronuclear (1H, 13C, 15N) Assignments
Backbone Experiments
Names of scalar
experiments based
on atoms detected
Consecutive residues!!
NOESY not needed
Heteronuclear (1H, 13C, 15N) Assignments
Backbone Experiments
CBCA(CO)NH
- inter-residue connectivity
(HN to previous Ca, Cb)
HNCACB
- intra-residue connectivity
(HN to own Ca, Cb)
Search 15N planes for 13Ca
and 13Cb chemical shifts
13Cb
R
H
H–C–H
H–C–H
chemical shift
H
H
H
O
H
13Ca
H
O
=
=
=
N–C–C–N–C–C–N–C–C
H
H
O
chemical shift
common 15N and HN chemical shift
in both experiments
(found on same 15N plane)
Heteronuclear (1H, 13C, 15N) Assignments
Backbone Experiments
CBCA(CO)NH
- inter-residue connectivity
(HN to previous Ca, Cb)
HNCACB
- intra-residue connectivity
and possibly inter-residue
(HN to own Ca, Cb)
Start with unique residue
1. Gly – only Ca
2. Ala – upfield-shifted Cb (~18
ppm)
3. Thr/Ser – downfield-shifted
Ca & Cb which are close to
each other
Heteronuclear (1H, 13C, 15N) Assignments
Side-chain Experiments
Multiple redundancies increase reliability
Heteronuclear (1H, 13C, 15N) 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 13C, 15N enrichment (also 2H)
High expression levels on minimal media
 Increased cost ($150/g 13C-gluocose; $30/g 15NH4Cl)
Structure Determination Overview
List of chemical
shifts for all
nuclei in protein
(1H, 13C, 15N)
NMR Experimental Observables
Provide Structural Information
1. Backbone conformation from chemical shifts (Chemical
Shift Index – CSI; Ha, Ca, Cb, 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 Ha, Ca, Cb, C’ determined chemical shifts
from protein to standard random coil chemical shift
values
• Upfield-shifted Ha and Cb and downfield-shifted Ca and
C’ values indicate amino acid residues in an a-helical
conformation (requires three consecutive residues
displaying this pattern)
• Downfield-shifted Ha and Cb and upfield-shifted Ca and
C’ values indicate residues in an extended (b-strand)
conformation
2. Hydrogen Bonds
C=O
• Slow rate of exchange
of labile HN with solvent
•Protein dissolved in
2H O; HN signals
2
disappear with time
•HN groups that are Hbonded (i.e. part of
secondary structure)
will exchange a lot
slower than those in
loops
H-N
3. Dihedral Angles from Scalar Couplings
•
•
• •
6 Hz
 Must accommodate multiple solutions multiple J values
4. 1H-1H 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
• 1H-1H NOESY
•
15N-
1H
or 13C dispersed 1H-1H NOESY
3D
4D
2D
1H
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  1H-1H distance (1/r6)
 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. 1H-1H 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?
Monitoring Molecular Interactions
15N-1H
HSQC
G27
NMR Provides
G22
A14
 Multiple probes
Y36
V19
 Site-specific
I24
S16
 In-depth info
V29
T23
M20
K33
F28
H31
W17
C37
L15
R25
A32
Q21
I34
D18
N30
S35
K26
 Spatial
distribution of
responses can be
mapped on
structure
Monitoring Molecular Interactions
Titration followed by 15N-1H 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/koff = t >> 1 sec  slow exchange, superposition of spectra
1/koff = t << 1 x 10-6 sec  fast exchange, weighted average
A
kon
koff
B
Kdiss = [A]/[B] = koff/kon
Ligand Binding
- Another protein
- Metal ion
- Drug or chemical
P + L = PL
Kdiss =
[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
Ptot = P + PL
Ltot = L + PL
So……. Kdiss =
[Ptot - PL] [Ltot - PL]
[PL]
Plot [Ltot]/[Ptot] vs “change” in NMR spectra
For fast exchange (weak binding):
Change =
obs - init
sat - init
=
[ PL]
[Ptot]
shifting of resonances in spectra
For slow exchange (tight binding):
Change =
Integral of peakobs
Integral of peakmax
=
[ PL]
[Ptot]
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
Rex < 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
15N
B
15N
15N
1H
1H
1H
• Same chemical shifts,
• Linewidth determined
• Fragments have
same structure
By size of molecule
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 HN solvent: slow timescales (milliseconds
to years!)
• requires local or global unfolding events
• HN 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 HN solvent: slow timescales (milliseconds
to years!)
• NMR relaxation measurements (ps – ns; ms – ms)
• R1 (1/T1) spin lattice relaxation rate (z-axis)
• R2 (1/T2) spin spin relaxation rate (xy-plane)
• Heteronuclear NOE (15N-1H)
Dynamics to Probe the Origin
of Structural Uncertainty

Weak correlation
Strong correlation 


- Measurements show if high RMSD is due to high flexibility (low S2)
NMR and Crystallography
NMR
X-ray
• Can mimic biological conditions
- pH, temp, salt
• Highly automated with more
objective interpretation of data
• information on dynamics
• Quality indicators (resolution, R)
• monitor conformational change
on ligand binding
• Surface residues and water
molecules well defined
• 2 structure derived from limited
experimental data
• Huge molecules and assemblies
can be determined
• need concentrated sample - lots
of protein; aggregation issues
• non-physiological conditions –
crystallization difficult
• size limited – ~40kDa for full
structure determination
• need heavy-atom derivatives –
production not always trivial
• more subjective interpretation
of data
• snap-shot of protein in time –
less indication of mobility
• lack of quality factors resolution and R-factor
• 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
Only NOEs from bound state
H
H
kon
koff
H