Practical Course in Protein NMR and EPR

1
Practical Course in Protein NMR and EPR
2006
2
Web-related stuff

• The powerpoints and handouts will be available
  at
• http//cetus.mchem.washington.edu
• Tons of web-related NMR resources can be found
  at
• http//cetus.mchem.washington.edu/labwiki/NEX/NEX1
  /nmr_resources.htm
• Web-related EPR resources can be found at
• http//cetus.mchem.washington.edu/labwiki/NEX/NEX1
  /epr_resources.htm

3
Course Plan

• Two Classes
• Day 1
• General Concepts in NMR
• 1D NMR
• Day 2
• 2D NMR and gt2D NMR
• CW and pulsed EPR

4
Purpose

• This is a practical course. Experimental design
  rather than theory.
• Qualitative treatment of NMR and EPR
• Classical view of magnetic resonance phenomenon
  rather than quantum mechanical

5
NMR/EPR

• NMR (Nuclear Magnetic Resonance)
• measurement of nuclear magnetic spin
• EPR (Electron Paramagnetic Resonance)
• measurement of electronic magnetic spin
• analogous to NMR but deals with electrons
• a.k.a. Also called ESR (Electron Spin Resonance)

6
Energy Levels
The bigger the magnet, the greater the separation.
Therefore, bigger magnets are able to get
higher resolution.
7
Magnetic Spin
If we spin at exactly 500 Mhz, the magnet will
appear to be standing still.
For a free proton in a 500 Mhz instrument,
the magnet is spinning at 500 MHz
Laboratory Frame
Rotating Frame.
Frequency of spinning is known as the Larmor
frequency
8
Chemical Shift

• ppm must be compared to a standard
• Why ppm?
• Most instruments are measured in Mhz.
• Gives whole numbers in terms of ppm.

9
Why do protons exhibit different chemical shift?
nuclei 1
nuclei 2

• Because they experience slightly different
  magnetic fields.
• Is it a big shift? No
• A nuclei that is found at 10 ppm on a 500 Mhz
  instrument has a frequency
• of 500.005 Mhz. NMR's have to be pretty precise
  indeed.
• Converting between Hz and ppm. A 1 ppm shift
  represents a 500 Hz on a
• 500 Mhz instrument.

10
Splitting of energy levels
observed
2 excited states

2 ground states
nucleus
splitting nucleus
11
Splitting of energy levels
observed

split by
splitting nucleus
nucleus
12
Splitting of energy levels
This is known as j-coupling
n2
n1
n2
n1
n1
coupled
uncoupled
13
Different Nuclei
proton
Gyromagnetic Ratio
C13
O17
N15
N14
-27 T-1 sec-1
19 T-1 sec-1
67 T-1 sec-1
-36 T-1 sec-1
268 T-1 sec-1
For labeled protein/drug
x 106
14
Different Nuclei with natural abundance
100
proton
Gyromagnetic Ratio
99.6
1
N15
O17
0.04
0.4
N14
C13
-27 T-1 sec-1
19 T-1 sec-1
67 T-1 sec-1
-36 T-1 sec-1
268 T-1 sec-1
For unlabeled protein/drug
x 106
15
Protons versus electrons
electron
Gyromagnetic Ratio
657 times bigger
proton
268 T-1 sec-1
176,086 T-1 sec-1
x 106
16
Relaxation
Z
Magnetic Field
Y
X
17
Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
ground state
18
Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
excited state
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Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
Because of thermal equilibrium the magnet will
want to return to the ground state via a spiral
path. You can think of the magnet as a top.
20
Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
Relaxation can be broken down into the x-y plane
and the z-axis
21
Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
Relaxation to equilibrium along the X-Y plane is
known as T2 relaxation or transverse relaxation.
22
Relaxation
Z
Magnetic Field
Y
X
Magnet spinning at the Larmor frequency (500 MHz
for a proton in a 500 MHz instrument.)
rotating frame
Relaxation to equilibrium along the Z axis is
known as T1 relaxation or longitudinal relaxation.
23
What are some things that affect T1 and T2
relaxation?

• temperature
• molecular weight
• viscosity of the solvent (e.g. 20 glycerol)
• paramagnetic centers (i.e. free electrons)

24
T1 and T2 versus Temperature
hot
cold
relaxation time
temperature
thick line T1 thin line T2
25
T1 and T2 versus Molecular Weight
small ligands (lt100 Daltons)
whole cells
large ligands (1000 Daltons)
small proteins (10 KD)
relaxation time
large proteins (100 KD)
MW
thick line T1 thin line T2
26
T1 and T2 versus Viscosity
no glycerol
add glycerol
relaxation time
viscosity
thick line T1 thin line T2
27
T1 and T2 relaxation versus distance from
paramagnetic center
24 Angstroms
relaxation
distance from paramagnetic center
Line T1/T2 relaxation
28
What is the effect of T1 and T2 on the NMR
spectrum?

• T1 and T2 relaxation are inextricably linked.
• A change in T1 will not cause an obvious change
  in the NMR spectrum.
• Shortening T2 will lead to a broadening of the
  NMR signal.

shorten T2
29
Chemical Exchange
nucleus of drug in protein
drug moving in and out of protein
nucleus from a drug in solution
protein
will experience a difference a
different magnetic field when it is in free
solution versus a protein
30
Chemical Exchange
nucleus in protein (bound)
nucleus in solution (free)
What happens when the rate of exchange is
increased?
31
Chemical exchange
intermediate exchange
fast exchange
slow exchange
intermediate exchange
Ross et al, 1984
Bound and free peaks mix
32
Saturation transfer
includes any transfer of magnetization of 1
nuclei to the next, ignoring the specific
mechanism (i.e. cross-relaxation)
transfer magnetization
unexcited nucleus
excited nucleus
unexcited nucleus
excited nucleus
33
Saturation Transfer NOE/ROE

• NOE and ROE have both specific mechanisms of
  saturation transfer known
• as cross-relaxation
• NOE (Nuclear Overhauser Effect) is saturation
  transfer along the z-axis
• ROE (Rotating Frame Overhauser Effect) is
  saturation transfer along the
• x-y plane
• What are their differences?

34
NOE and Molecular Weight
1000 Daltons
0.5
NOE
0
-1
Molecular Weight
35
ROE and Molecular Weight
1
0.5
ROE
positive for all values of MW
0
Molecular Weight
36
Saturation Transfer Spin Diffusion

• occurs when saturation transfer through multiple
  nuclei.
• can lead to erroneous NOE's or ROE's. Correct
  NOE's and ROE's rely on interaction between pairs
  of nuclei.
• must always keep this in mind when considering
  saturation transfer.

37
What happens to the NMR signal when saturation
transfer occurs?
Saturation transfer
or
to the nuclei corresponding to the peak.
peak can get bigger or smaller
38
Basic NMR Concepts Summary

• Energy Levels
• Magnetic Spin
• Chemical Shift
• Splitting of Energy Levels (j-coupling)
• Different Nuclei
• gyromagnetic ratio
• natural abundance
• Relaxation
• T1 relaxation
• T2 relaxation
• Chemical Exchange
• Saturation Transfer
• NOE/ROE
• Spin Diffusion

39
1D-NMR experiments
40
Why is concentration important?

• relationship between concentration and signal
  amplitude (signal-to-noise) is linear
• relationship between average and signal-to-noise
  is squared.
• e.g. If we half the concentration of a sample
  that takes 9 hours to average, we will need to
  run the experiment 81 hours to get the same
  signal-to-noise. On the other hand, if we double
  the concentration, that same experiment will take
  only 3 hours to get the same signal-to-noise.

41
1D-Proton
What concentration do I need? 100 mM
minimum What solvent do I have it in? D2O
You run the experiment and you see only 1 peak at
5 ppm.
e.g.
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Solvent Suppression
That 1 peak is probably water. You need to
suppress that water signal. There are lots of
ways to do it. 2 of the most common are

• presaturation
• WATERGATE (WATER suppression by GrAdient Tailored
  Excitation)

They work by two opposing mechanisms
presaturation works be saturating the water
signal WATERGATE works by exciting everything
but the water signal.
43
Presaturation

• requires that you excite a given peak for an
  extended period of time before the acquiring
  pulse.
• results in equal populations of excited and
  ground nuclei.
• Therefore, no signal.
• Caveats presaturation can lead to spin diffusion.

44
WATERGATE

• works on a different strategy than presaturation.
• excite everything, but the water. Excited areas
  are shown in blue.
• CAVEATS requires special instrumentation.
  Requires more complicated pulse sequence. May
  kill signal near water.

45
Example of 1D proton using watergate
water signal after watergate suppression
46
Unlabeled 1D Proton Why not protein?

• Too many signals
• T2 relaxation too fast signals broaden

very broad signal
protein
T2
ppm
MW
47
Unlabeled 1D Proton Why not protein?

• Ways to deal with it?
• isotopically label specific parts of the protein
• use special pulse sequences
• use higher field NMR to separate the broadened
  peaks.

48
1D Proton Protein/Drugs and Chemical Exchange
Because of chemical exchange, the actual
1D proton spectrum can provide information about
drug kinetics.
drug bound
drug free
49
1D Proton Protein/Drugs and Chemical Exchange
ideal situation
In this case, half of ligand is bound to protein
and half is free. The protein concentration
would be half the drug concentration in this
case.
drug bound
drug free
50
1D Proton Protein/Drugs and Chemical Exchange

• need to minimize background protein signal
• use low protein concentration/high drug
  concentration

free drug
bound drug (short T2)
background protein signal
ppm
51
1D Proton Protein/Drugs and Chemical Exchange

• What is the effect of high ligand to protein
  ratios on the chemical exchange NMR signals?

protein (fast exchange)
protein (slow exchange)
protein (intermediate exchange)
no protein
52
1D Proton Protein/Drugs and Chemical Exchange

• Only fast exchange will show a noticeable change,
  when protein concentration is low. (i.e. shift in
  peak)

protein (fast exchange)
protein (slow exchange)
protein (intermediate exchange)
no protein
53
1D Proton Protein/Drugs and Chemical Exchange

• This is why a lot of NMR techniques involving
  protein and ligands must be fast exchange.

protein (fast exchange)
protein (slow exchange)
protein (intermediate exchange)
no protein
54
1D-C13
What concentration do I need?
The minimimum concentration for proton is 100 mM.
Gyromagnetic ratio of a proton is about 4 times
larger than carbon. Therefore, we need to have 4
times the concentration in order to get the same
signal. 100 mM 4 400 mM
That doesn't look too bad. However, if we have an
unlabeled sample, we must also consider natural
abundance. For C13, it is about 1. In order to
equal the concentration of 100 mM protons, we
must multiply by 100.
Thus 40 mM is the minimum concentration we need
for 1D-C13
400 mM 100 40 mM
55
T1-relaxation measurement

• determine time for an excited nucleus to reach
  equilibrium along the z-axis.
• previously, we talked about the affect of a
  paramagnetic center on T1 relaxation.

closest distances predominate
T1
r6 relationship
distance to paramagnetic center
56
T1-relaxation measurement

• Used to examine the interaction between the
  paramagnetic heme of Cytochrome P450 (P450)and a
  bound drug.
• Method
• Step 1 Measure T1 of drug with oxidized P450
• Step 2 Measure T1 of drug with CO bound to
  reduced P450
• Oxidized P450 will have both paramagnetic and
  diamagnetic contributions.
• Reduced P450 will only have diamagnetic
  contributions.
• To find out the paramagnetic contributions, we
  need to take the difference.

57
T1-relaxation measurement
difference in T1 from the first two experiments
58
T1-relaxation measurement
What other factors come into play with T1
relaxation?
59
Other factors that come into play with T1
relaxation experiments

• Correlation time (i.e. Molecular Weight,
  Viscosity, Temperature)
• Spin state (high spin and low spin)
• Major Assumptions
• the CO-reduced P450 binds drug the same as
  oxidized P450.
• that the electrons are localized to a point at
  the Fe in the heme
• spin state is homogeneous either all high spin or
  all low spin

60
Other factors that come into play with T1
relaxation experiments

• Because of potential interference from the
  protein signal, experiments are carried out with
  ligand to protein ratios of 10,000 to 1.
  Therefore, ligands must be fast exchange, since
  other exchange regimes will not be observable.
• What are a couple of methods to determine
  exchange regime?

61
Determining exchange regime
Method 2 Temperature dependence of T1
Method 1 Look for peak shift
T1
plus protein
minus protein
temperature
Positive slope fast exchange
Problematic because of spin state temperature depe
ndence
62
Example of a T1 relaxation experiment
63
Resulting Model
drug
heme
Heme hover model
64
STD

• STD (Saturation Transfer Difference)
• Method to look at protein-drug interaction by
  examining the saturation transfer from the
  protein to the drug.

65
STD mechanism
protein
unexcited drug
66
STD mechanism
protein
unexcited drug
Protein excited by spin diffusion.
67
STD mechanism
Nuclei on the drug are excited differentially,
allowing for one to better under stand drug
protein interaction
protein
Saturation transfer to the drug via NOE
mechanism or other mechanism
excited drug
68
Relationship between STD and KD
weak binders don't get much saturation from the
protein
tight binders relax before exchange
observed STD amplitude
1000
1
100
10
0.01
0.1
KD (mM)
69
STD

• Allows for mapping of drug protein interaction.
• Pro's
• can be done with minute amounts of protein
• Con's
• must be fast exchange (must not bind too tight)
• doesn't work well with proteins that are in a
  paramagnetic state (e.g. oxidized P450)

70
STD example
Mayer, et. al 2001
71
Waterlogsy

• Waterlogsy Water ligand observed via gradient
  spectroscopy
• Can provide KD's
• Can provide information about the aqueous
  environment of a ligand.

72
Waterlogsy
free drug
protein bound drug
protein
unexcited water
unexcited drug
73
Waterlogsy
protein
water excited by spin diffusion
excited water
unexcited drug
74
Waterlogsy
protein
water transfers saturation by NOE mechanism to
the drug.
unexcited water
excited drug
75
Waterlogsy
waterlogsy signal is an average of bound and free
NOE enhancement
water to free ligand NOE
0.5
NOE
water to bound ligand NOE
-1
Molecular Weight
76
Waterlogsy
0.5
NOE
0
Tight KD mostly bound to protein
-1
KD
77
Waterlogsy
weak KD mostly free
0.5
NOE
0
-1
KD
78
Waterlogsy
0.5
NOE
0
-1
KD
79
Waterlogsy
1
waterlogsy signal
0
-0.5
KD
80
Waterlogsy
1
without chemical exchange considerations
waterlogsy signal
0
-0.5
KD
1
mM
10
100
1000
10,000
1
Tight binders relax before exchanging
with chemical exchange considerations
waterlogsy signal
0
-0.5
1
10
100
10,000
1000
KD
mM
81
Waterlogsy Pro's and Con's

• Pro's
• provides information about the aqueous
  environment
• can differentiate between relatively tightly
  bound and weakly bound by a change from positive
  to negative amplitude.
• possible to measure KD
• Con's
• must be fast exchange
• very complicated pulse sequence
• signal is weak compared to STD, despite authors
  comments

82
Waterlogsy Example
(Dalvit et al, 2002)
83
1D NMR Experiments Summary

• Concentration?
• 1D-proton
• solvent suppression
• presaturation
• WATERGATE
• 1D-C13
• T1 relaxation for P450
• relation of distance and T1
• determining exchange regime
• example
• STD
• mechanism
• Pro's and Con's
• KD vs STD
• example

84
1D NMR Experiments Summary (continued)

• Waterlogsy
• mechanism
• KD vs. Waterlogsy
• Pro's and Con's
• Example

85
References
NMR
1. Macomber, R. S. (1998) A Complete Introduction
to Modern NMR Spectroscopy, John Wiley Sons,
Inc., New York. Comments Very good and easy
introduction to NMR. Very simple mathematics.
Lacks modern use of gradients. Also,
discusses continuous wave-EPR. 2. Claridge, T.
D. W. (1999) High-Resolution NMR Techniques in
Organic Chemistry, Vol. 19, 1st ed., Pergamon,
New York. Comments This reference goes from
beginning to advanced NMR. Very simple and easy
to understand mathematical explanations. Discusses
NMR field gradients and modern NMR techniques.
This is one of the best references in NMR.
Explains some pratical aspects of
NMR 3. Levitt, M. H. (2001) Spin Dynamics
Basics of Nuclear Magnetic Resonance, John Wiley
Sons, LTD, New York. Comments A very good
mathematical introduction into NMR. Fairly
simple explanation of very difficult
concepts. 4. Berger, S., Braun, S., and
Kalinowski, H.-O. (2004) 200 and More NMR
Experiments A Practical Course, Wiley, John
Sons, Incorporated. Comments Good for all
levels of expertise. Best practical book on NMR.
This book is a good starting point for any
experiments. 5. Neuhaus, D., and Williamson, M.
P. (2000) The Nuclear Overhauser Effect in
Structural and Conformational Analysis,
Wiley-VCH. Comments For advanced users that
want to use NMR for structural determination.
86
References
T1 relaxation
1. Solomon, I., and Bloembergen, N. (1956)
Nuclear magnetic interactions in the HF molecule,
The Journal of Chemical Physics 25,
261-266. 2. Regal, K. A., and Nelson, S. D.
(2000) Orientation of caffeine within the active
site of human cytochrome P450 1A2 based on NMR
longitudinal (T1) relaxation measurements, Arch.
Biochem. Biophys. 384, 47-58. 3. McLaughlin, A.
C., and J. S. Leigh, J. (1973) Relaxation times
in systems with chemical exchange approximate
solutions for the nondilute case, J. Magn. Reson.
9, 296-304. 4. Pintacuda, G., Hohenthanner, K.,
Otting, G., and Muller, N. (2003) Angular
dependence of dipole-dipole-Curie-spin
cross-correlation effects in high-spin and
low-spin paramagnetic myoglobin, J. Biomol. NMR
27, 115-132.
STD
1. Claasen, B., Axmann, M., Meinecke, R., and
Meyer, B. (2005) Direct observation of ligand
binding to membrane proteins in living cells by a
saturation transfer double difference (STDD) NMR
spectroscopy method shows a significantly higher
affinity of integrin alpha(IIb)beta3 in native
platelets than in liposomes, J. Am. Chem. Soc.
127, 916-919. 2. Gharbi-Benarous, J., Bertho, G.,
Evrard-Todeschi, N., Coadou, G., Megy, S.,
Delaunay, T., Benarous, R., and Girault, J. P.
(2004) Epitope mapping of the phosphorylation
motif of the HIV-1 protein Vpu bound to the
selective monoclonal antibody using TRNOESY and
STD NMR spectroscopy, Biochemistry 43,
14555-14565. 3. Mari, S., Serrano-Gomez, D.,
Canada, F. J., Corbi, A. L., and Jimenez-Barbero,
J. (2004) 1D saturation transfer difference NMR
experiments on living cells the
DC-SIGN/oligomannose interaction, Angew. Chem.
44, 296-298. 4. Shimotakahara, S., Furihata, K.,
and Tashiro, M. (2004) Application of NMR
screening techniques for observing ligand binding
with a protein receptor, Magn. Reson.
Chem. 5. Mayer, M., and Meyer, B. (2001) Group
epitope mapping by saturation transfer difference
NMR to identify segments of a ligand in direct
contact with a protein receptor, J. Am. Chem.
Soc. 123, 6108-6117. 6. Wang, Y. S., Liu, D., and
Wyss, D. F. (2004) Competition STD NMR for the
detection of high-affinity ligands and NMR-based
screening, Magn. Reson. Chem. 42, 485-489.
87
References
Waterlogsy
1. Dalvit, C., Pevarello, P., Tato, M., Veronesi,
M., Vulpetti, A., and Sundstrom, M. (2000)
Identification of compounds with binding affinity
to proteins via magnetization transfer from bulk
water, J. Biomol. NMR 18, 65-68. 2. Dalvit, C.,
Fogliatto, G., Stewart, A., Veronesi, M., and
Stockman, B. (2001) WaterLOGSY as a method for
primary NMR screening practical aspects and
range of applicability, J. Biomol. NMR 21,
349-359. 3. Dalvit, C., Fasolini, M., Flocco, M.,
Knapp, S., Pevarello, P., and Veronesi, M. (2002)
NMR-Based screening with competition water-ligand
observed via gradient spectroscopy experiments
detection of high-affinity ligands, J. Med. Chem.
45, 2610-2614. 4. Shimotakahara, S., Furihata,
K., and Tashiro, M. (2004) Application of NMR
screening techniques for observing ligand binding
with a protein receptor, Magn. Reson. Chem.