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Outline of NMR Spectroscopy
Lecture one
A.
B.
NMR Basics
I.
NMR Phenomenon-Nuclear Spin
II.
Relaxation
III.
J coupling
IV.
Dipolar coupling
V.
The nuclear Overhauser effect (NOE)
VI.
Chemical shift
Molecular Motion and kinetics
I.
The NMR timescale
II.
Exchange
III. Molecular motion
NMR
N uclear
M agnetic
R esonance
NMR takes advantage of an intrinsic dipole moment some
nuclei have.
Why do some nuclei have an intrinsic dipole
moment and other do not?
If a nucleus has an uneven distribution of charge, this
induces a magnetic field, called spin. This nucleus behaves
like a little magnet.
In general: if the number of protons or neutrons is odd, then
the spin is a multiple of 1/2 (i.e. 1/2, 3/2, etc.). If both are
odd, then the spin is 1. If both are even, then the nuclei has
no spin-it has an even distribution of charge and is not
magnetic.
Atoms are little magnets
Induced magnetic field
Nuclear cloud
These little magnets affect each other as a function of
DISTANCE to each other.
The longest distance that can be “seen” from one atom to
another atom is ~5 A.
Classic representation of a spin
Bo
+1/2
The spin precesses in the
induced magnetic field, Bo
-1/2
Spin motion
Bo
Spins in field
Table of NMR active Nuclei
Nuclei
Spin #
Natural Abundance, %
1H
1/2
99.98
2H
1
0.015
12C
0
98.9
13C
1/2
1.1
14N
1
99.62
15N
1/2
0.4
16O
0
99.76
17O
5/2
0.039
19F
1/2
100
23Na
3/2
100
31P
1/2
100
59Co
7/2
100
What a spectrometer does
Sample
Solenoid
Liquid He dewar
Liquid N2 dewar
Air Cushions
Probe
Energy and wavelength
M
100 10
mm
mm
1
100 10
1
100 10
nm
1
100 10
1
g, wavelength
n, energy
3*106
3*108
NMR
3*1010
Microwave
Radio waves
IR
3*1012
3*1014
VIS
UV
3*1016
X-Ray
g-Ray
Frequency of Nuclei
The frequency at which a given nucleus precesses about the
induced magnetic field, Bo , is given by the Larmor equation:
n= g/2p |Bo|
Where g is the gyromagentic ratio for the given nucleus and Bo
is in hertz. This is the Larmor frequency (of that nucleus).
For example, when someone tells you that they have a 600
MHz NMR instrument, they are telling you the Larmor
frequency of the proton nucleus for that magnet.
Chemical Shift
Chemical Shift is the most basic parameter of NMR and is
defined as d in parts per million (ppm).
d= (w-w0)/w0 x 106
Where w0 is the Larmor frequency in Hz of the reference line
and w is the resonant frequency of the line.
The chemical shift of a nucleus originates in the electron
cloud around a nucleus, which induces a local magnetic field
which opposes the applied field. This leads to the term
shielding and deshielding. The more shielded a nucleus is,
the larger the chemical shift and conversely, the more
deshielded a nucleus is, the smaller the chemical shift.
Shielding
K. Wuthrich, “NMR of Proteins and Nucleic Acids”.
Typical Chemical Shifts
Protein chemical shifts
DNA and RNA chemical shifts
Relaxation
Relaxation is the precession of the spin from the perturbed
state (x or y) to the relaxed state (z ).
There are two ways nuclei do this:
1. Spin-lattice or longitudinal relaxation, T1, which returns
magnetization to equilibrium.
2. Spin-spin or transverse relaxation, T2, which leads to loss
of phase coherence and signal.
90o pulse
z
90o pulse, B1
y Detector
x
Detector
The Rotating
Frame
T1
z
Bo
y
x
Mz = Mo(1-e -t/T1)
T1 is the time constant at which the magnetization returns to equilibrium
(when all of the magnetization is aligned along z).
T1 is experimental determined many ways, but the most common is the
inversion recovery method. In structural work, T1 is estimated based on
the molecule’s molecular weight (discussed in Molecular motions).
5xT1 is the time for return of >99% of magnetization to equilibrium.
This the amount of waiting time between experiments.
T2
T2 is the spin-spin relaxation.
T2 represents the difference in magnetization between otherwise
identical nuclei. T2 has to due with sample inhomogenity and this
contributes to line broadening. A simple approximation of T2, T2*:
n1/2= 1/pT2*,
where n1/2 is the linewidth at half height.
z
Many different conformations for
identical spins, causing slight
variations in chemical shift.
y
x
Peak width at half height
n
n1/2
d, Hz
Exchange
When a nucleus is exchanging between two or more
different environments, this will give rise to complicated
spectra.
Exchange is important in structural work when you are
looking at a dynamic system.
For example, if you have a side chain that is moving
slowly on the NMR timescale, between two different
environments, it can give rise to multiple peaks that can be
described by T2.
J or scalar coupling
Most common coupling between two or more adjacent spin
½ nuclei.
The general formula for splitting is:
# of lines in the multiplet (N)= 2nI + 1, where I is the spin of
the neighboring nuclei and n is the number of identical
nuclei. When I=1/2, this simplies to N=n+1.
In structure work, J coupling is critical to measuring the
bond angles and dihedral angles between nuclei. The
Karplus equation is used to calculate the dihedral angles for
3 bond or vicinal coupling:
J = A + B cosq+ C cos2q
Where A, B, C are coefficients that depend on the nuclei
electronegativity.
Ethanol – A Classic NMR 1 D experiment
H
H
H
C
C
H
H
O
H
TMS
d, ppm
0
Dipolar coupling
Dipolar coupling is the interaction between two dipoles
(of two different nuclei) who are not connected through a
bond.
This is one of the most important ways nuclei relax. It
contributes the most to T1 and is heavily dependent on
molecular motion of the molecule.
Nuclear Overhauser Effect
The NOE is the transfer of magnetization between nuclei. This is the
most important effect in structural work.
It is important to note that it is heavily dependent on molecular motion
(tc) and distance (rIS). A general equation that describes the NOE:
R1 = K g2I g2S rIS-6 tc,
where I and S are the interacting nuclei.
NMR timescale
NMR has a broad time scale in terms of what it can
“see”.
The useful range for structural analysis are from
seconds to microseconds.
Anything much faster than microseconds, the event
tends to be averaged out and a single event is seen.
Much slower than seconds leads to line broadening (a
T2 effect) and decreasing peaks (NOE effect), which
lead to the reduction and sometimes disappearance of
the signal.
Some useful timescales
Average chemical reaction: ns (ps to ms)
Average enzymatic reaction: ms (s to ns)
Average tumbling time for a globular protein: 107 Hz
or 100 ns
Average tumbling time for a small molecule: 1011 Hz
or 10 ps
Molecular motion
NMR is very dependent on how molecules rotate in solution. It can be
calculated using the Debye equation (assuming a spherical shape):
tc= 4pha3/3kT
h=viscosity of the solvent and a=radius of the molecule. An
approximate tc, assuming typical values for organic solvents, is:
tc =10-12 Mw, where Mw is in Daltons.
Since this isn’t always the case, relaxation rates (T1 and T2) and
NOE measurements can be used to calculate the correlation time.
Normally, this is done through other means (like fluorescence
anisotropy or DLS).
Reference Signal
All NMR spectra must be referenced.
This means that there has to be a compound that has a known
chemical shift in the sample.
For example, in organic chemistry NMR, TMS
(tetramethylsilane) is used to reference spectra-it is set to
zero ppm.
For structural work, the reference is set to the most common
molecule/frequency.
Reference Table
Isotope
Detected
1H
Reference
Compound
H2O
Chemical
Shift, ppm
4.7
15N
NH4Cl
125.7
13C
Glucose
50.5
17O
H2O
0.0
31P
H3PO4
0.0
Data Processing
Raw Data
t
Fourier transformation
Processed data
Lecture 2 Outline
I.
Multidimensional NMR
A.
B.
Why do we need
Multidimensional NMR?
2 D NMR
1.
Nomenclature
2.
Basic experiments
a.
COSY
b.
NOESY
c.
TOCSY/HOHAHA
II.
C.
3 and 4 D NMR
1.
Why we need 3 and 4 D NMR
How to make a multidimensional
experiment
3.
How to read a 3 and 4 D
experiment
Structural Information and Structure
Generation
A.
Distance Measurements
B.
Coupling Constants and Angle
measurements
C.
Energy Minimization and
Restrained Molecular Dynamics
D.
Quality of NMR structures
d.
3.
Multiple Quantum
Filtration
Solvent supression
2.
How to get a protein structure by NMR
Clone and express lots of soluble protein
Preliminary Spectra (1D 1H, NOESY,
COSY)
Sequential assignment
Collection of conformational contraints
Calculation of 3D structure
Protein Solutions
Not just any protein structure can be solved by NMR
If the protein meets these conditions, then it’s a good chance
it’s structure can and will be solved by NMR:
1. Mw range: up to 30 kD (for most practical purposes)
2. Soluble at high concentrations (0.5- 1.0 mM) with no
aggregation (need to rotate freely)
3. Must be able to purify to 95% or greater
4. Stable at room temperature for days/weeks. Better if
relatively temperature insensitive.
5. Must be structured on the NMR timescale
Protein Considerations
Up to 15-18 kD, it is possible to do the structure without
isotopic labeling
From 15- ~30 kD, need 13C/15N labeling in the protein
From 30 kD and up, not only need 13C, 15N labeling,
also need 2H labeling
Above 40 kD, need either solid state NMR or special
NMR techniques to remove tc requirement in NOE (and
other parameters)
Isotopic labeling
In order for proteins to be isotopically labeled, they must be grown in
the presence of isotopically labeled nutrients.
This requires some knowledge of metabolism of E. coli or the
organism used to produce the labeled protein
In the case of E. coli, a strain is used that can grow on minimal
media. (i.e. BL21, DH5a)
The cells are grown in minimal media made with uniformly labeled
15N ammonium chloride or 13C glucose.
Protein purification proceeds as without labeling
Why Multidimensional NMR?
Multidimensional NMR helps resolve resonances that would
otherwise be overlapped and not identifiable.
The multidimensional component refers to another frequency
(not time, space, etc.)
We need to further resolve spectra in biological NMR because
our molecules are not simple.
Typical Chemical Shifts
Protein chemical shifts
DNA and RNA chemical shifts
1 D NMR Spectra
1H
1D NMR of Lysozyme
1H
1D NMR of DNA
Interesting Facts
In a protein/peptide:
There are at least 20 spin systems (1 for each
amino acid)
The average number of protons per amino acid is 8
There are a maximum of ~20 inter-residue contacts per
amino acid (in a structured protein/peptide)
In DNA/RNA:
There are 4-5 spin systems (1 for each base)
The average number of protons per base is 11
There are a maximum of ~80 inter-residue contacts per
base
Experimential design flow chart
2 D NMR- Nomenclature
NMR experiments are named in acronyms
Some examples:
COSY – COrrelation SpectroscopY
NOESY- Nuclear Overhauser Effect SpectroscopY
TOCSY- TOtal COrrelation SpectroscopY
HOHAHA-HOmonuclear HArtman HAhn
QF- Quantum Filtered
Protein Connectivities
Short range
Longer range
2 D NMR- Basic Experiments
Staple experiments for structural work
1. COSY (1H, 1H)
2. NOESY (1H, 1H)
3. TOCSY/HOHAHA (1H, 1H)
These are the basic experiments that form the framework
of more specific experiments
COrrelation SpectroscopY
COSY experiments show who is coupled to who (dipolar coupling and J
coupling)
This experiment:
Confirms the sequence of a protein
Sees residues that are coupled together (very close together)
This basic experiment can be used in many different ways to look at
different couplings (i.e. 1H-13C coupling) and to filter out couplings
(quantum filtering)
COSY in the rotating frame
z
90o pulse
90o
pulse, x
y
x
t1
Detector
This is for a one spin system
COSY-spectrum
R
N
Ca
H
H
O
C
1H
1H
COSY of Nucleic Acids
K. Wuthrich, “NMR of Proteins and Nucleic Acids”
COSY of Amino Acids
K. Wuthrich, “NMR of Proteins and Nucleic Acids”
Quantum Filtering
A quantum is a single resonance (also known as Single
Quantum, SQ).
Double/Triple/Multiple Quantum (DQ, TQ and MQ) are
multi-coupled resonances.
With filtering, you are selecting for the quantum number
so named and is useful when 2 or more step
connectivities are required.
i.e. DQF-COSY selects for resonances that are coupled
twice (hence, Double Quantum).
TOtal Correlated SpectroscopY
Otherwise known as HOmonuclear HArtman HAhn
TOCSY experiments detect indirect J coupling though crosspolarization rather than by relayed magnetization transfer
(relayed magnetization transfer experiments are called RELAYCOSY)
Cross-polarization (which is caused by spin-locking) causes the
spins to become equivalent and give rise to a single resonance.
The time it takes for this to happen is equal to 1/2J
This experiment is repeated at various spin-lock times in order to
measure this coupling constant.
The difference between TOCSY and HOHAHA is in how the
spin-locking is applied, but these experiments yield the same
information
TOCSY-spectrum
H
N
Ca
H
H
O
C
1H
1H
Heteronuclear Correlation Spectroscopy
These experiments are also known as HMQC and HSQC
(Heteronuclear Multiple/Single Quantum Coherence)
This experiment is essentially the same as a COSY, except the
magnetization transfer is to another coupled nucleus at another
frequency
This is done simultaneously with the (usual) proton pulses
Homonuclear coupling can interfere with the effect you are
trying to observe, so 1H-1H coupling is irradiated (decoupled)
The delay in this experiment measures ¼ JIS
Heteronuclear couplings are usually very weak effects
Nuclear Overhauser Effect SpectroscopY
NOESY experiments detect NOEs (magnetization transfer between 2 or more nuclei)
This experiment can “see” other nuclei up to 5 A away through space
NOESY will give the same information as a COSY, plus the through space
information
If a standard distance is known and NOEs can be measured, the NOE equation can be
simplified to:
rIM/rIS = (aIS/aIM)1/6
Where r is the distance between IM and IS and a is the crosspeak intensity
Also for this to be true, NOE buildup curves must be done in order to be assured that
you are at maximum NOE intensity
NOESY in the rotating frame
z
90o pulse
y
tm
90o
pulse, x
x
t1
Detector
This is for a one spin system
NOEs for DNA
Solvent Suppression
Very necessary in biological NMR since our samples are in 1H2O.
Solvent suppression involves irradiation of the water signal with
high power irradiation. This saturates the signal by equalizing the
populations of + and – spin states.
This can be avoided by putting the sample in 2H2O, but for protein
samples, this is not easily accomplished.
Pulse sequences
3 D and 4 D NMR
Why do we need 3D and 4D experiments?
2D experiments can get very crowded as the number of
residues increases
3 and 4D experiments can help resolve spectral overlap by
modulating 2 D experiments in various time domains which
lets different magnetization buildup (and selecting against
other magnetization)
3 and 4 D experiments
How to read a 3 or 4 D experiment
2 D to 3 D
2 D to 3 D to 4 D
Structural information
Experiments are finished, how do we compile the
data?
How data is fitted
Energy Minimization and
Restrained Molecular Dynamics
This subject is a whole class unto itself.
These computational techniques are used to evaluate
the model generated by the data, then help correct
errors via minimization and dynamics.
Modeling is frequently used for sidechains, even
residues, that cannot be visualized by NMR (motion
is too rapid/slow).
Because the data typically gives a range for distances
and angles, multiple structures are generated.
The more structures generated the better.
Quality of NMR Structures
Since a lot of structures are generated, one quick quality
assessment is the RMSD between them.
The RMSD should not be more than 2 A
The RSMD equation (for any structure comparison):
RMSD = (1/N S (ri – r’i)2)1/2
Where N is the number of atoms being compared, r is the
atomic coordinates for the structures in question
Another quality assessment for NMR structures is the
number of NOEs per residue (both intra/inter residue
NOEs)
NOEs per Residue
A general rule for resolution of NMR structures (to
keep nomenclature consistent with Crystallography:
Less than 5 NOEs per residue = 8 –10 A structure
6-12 NOEs per residue = 5 A structure
12-15 NOEs per residue = 3 A structure
16-20 NOEs per residue = 2.3-2.5 A structure
Over 21 NOEs per residue = 2.0 A structure
Clore GM and AM Groenborn (1991) Science
Dynamics
This is NMR’s real strength. Can tell what atoms are
mobile and how fast they vibrate*.
In order the evaluate dynamics in NMR, one needs to
measure T1, T2 and tc.
After these values are obtained, an order parameter is
calculated (S2).
An order parameter is calculated for all atoms (got the
information, why not?) and plotted. In an average molecule
there is motion, but mobile regions are visible by large
shifts in the order parameter compared to the backbone
atoms.
Homework Handout
Question 1.
Nucleus
Spin Number
Natural
Abundance, %
1
1-0 odd 1/2
99.98
2
2-1 even, odd 1
0.015
12
12-6 even, even 0
98.9
13
13-6, odd, even 1/2
1.1
14
14-7, even, odd 1
99.62
15
15-7, odd, odd 1/2
0.4
16
16-8, even, even 0
99.76
17
17-8, odd, even 5/2
0.039
19
19-9, odd, odd 1/2
100
23
23-11, odd, odd 3/2
100
31
31-15, odd, odd 1/2
100
59
59-27, odd, odd 7/2
100
H
H
C
C
N
N
O
O
F
Na
P
Co
Question 2
2A. Without isotopic enrichment, which of the nuclei above
would be found in significant levels in biological systems?
1H, 12C, 14N, 16O, 23Na, 31P
2B. How would you enrich a particular protein with an isotope
that isn’t at a biologically significant level?
To enrich a protein with an isotope, it would need to be
produced in the presence of the isotope in question: Cells
would need to be grown in the presence of compounds
enriched for the isotope(s).
Question 3
A.
NMR
X-ray
Solvent conditions more physiological Solvents- uses a lot of
detergents, salts
Nondestructive
Destructive
Sees protons, connectivities first,
Sees overall physical structure
then model physical structure
first, have to model in protons,
connectivities
Can take months, sometimes years
Takes minimum 1 month, up to
a year
Can use as little as 0.1 mM to 1 mM
Needs several mM of protein to
screen for crystals
Can be used to study dynamics
Motion is bad
Question 3B
B. Because of their differences, they can be used to compliment one another,
however it has to be certain that the solvent conditions did not change the overall
structure of the molecule. Small changes can be accommodated, but large ones
will cause contradictions.
1. NMR can confirm that crystallization doesn’t give a structure due to solvent
conditions.
2. NMR can help solve the phase problem for crystallography, but 1 has to be true
first.
3. If a region appears to be mobile in the crystal, NMR can confirm this and get a
minimized structure of the mobile region to complete the picture.
4. If working together, a relatively large protein (25-40 kD) can be solved fast.
Problems in the book – 12.2
Consider spin-spin (through bond) splitting of proton peaks
for CH3-NH2. A. Diagram the possibilies for the various
interactions. B. Show the spectrum with the expected
splittings and relative intensities.
H
H
C
H
N
H
H
ppm
Question 12.5
Given that Glu has the following peaks: NH=8.4, aH=4.3, bH=2.1 and 1.9, gH=2.3 for
both; at what 2D coordinates do you expect COSY interactions?
-OOC
HN
H
H
Ca
Cb
Cg
H
H
H
O
Cd
O-
1H
1H
Remember: The knowing the exact chemical shift is not necessary, but knowing the pattern
and what each peak represents is!
Question 12.6
In the NOESY below, the off-diagonal circles are crosspeaks that were in
the corresponding COSY, while the crosses are new crosspeaks in the
NOESY. Explain, using proton numbers, what each crosspeak tell us about
the molecule.
Things to remember:
Diagonal are crosspeaks to themselves
Peaks that are in the COSY, but not NOESY are
correlated to each other
New Peaks in the NOESY tell us about non-coupled,
spins that are close in space.
Question 12.6-NOESY
Cell paper
“Inhibiting HIV-1 Entry: Discovery of D-Peptide Inhibitors that
Target the gp41 Coiled-Coil Pocket
Eckert, DM et al. Cell (1999) 99:103-115
What did they do: Discovered a pocket on HIV-1 gp41 and exploited it by
designing D-peptides that bind in it. They appear to make good contact and
are therefore good inhibitors of HIV-1 infection. They used a variety of
structural techniques to confirm binding and contact of the peptides with the
pocket.
What did they use NMR for? They used NMR to confirm they mode of
binding. Specifically, they used NMR to show a shift in the trp571
resonances, which indicate that trp571 is involved in binding of the peptides.
This is a good example of the use of shielding!
Figures from Cell paper
Structural characterization of the complex of the Rev
response element RNA with a selected peptide
Zhang, Q. et al. Chemistry and Biology (2001) 8: 511-520
What did they do: Did the structure of the RSG-1.2 peptide in
complex with the Rev response element RNA because its
sequence is different than the natural Rev peptide and therefore
its binding should be different than Rev.
NMR conditions:
Peptide = 19 residues, RNA = 85 bases
They used both unlabeled samples and 13C/15N samples
They also dissolved their samples into D2O
Used 15, 25, 35 and 45 oC for their experiments
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
How good is it?
This works out to be: 189 restraints/19 residues = ~10
restraints/residue for the peptide
For RNA: 620 restraints / 84 bases = ~7 restraints/base
These RMSD measurements are for the converged structures, NOT the
structure(s) generated by NMR!
Questions to think about
1. Why did they label both the peptide and the RNA? The
peptide is small (only 19 residues, Mw ~20Kd)…
2. Why different temperatures? Why would that help?
3. Do you believe this structure? Why or why not?
4. How would you make this better (theoretically, of course)?
“Solution structure of cyanovirin-N, a potent
HIV-inactivating protein”
C.A. Bewley, et al.
Nature Structural Biology (1998) 5:571-578
“The Domain-Swapped Dimer of
Cyanovirin-N is in a Metastable Folded
State: Reconciliation of X-Ray and NMR
Structures”
L. G. Barrientos, et al.
Structure (2002) 10: 673-686
What is cyanovirin-N?
A protein isolated from the cyanobacterium, Nostoc ellipsosporum.
Identified by a general screening effort to find products with antiviral/HIV activity
What does it do?
Unknown function in cyanobacterium
But…is a very potent inhibitor of HIV
How does it work?
It prevents fusion of the virus to the host cell (helper T4 cells) by
binding to gp120 on the viral surface envelope.
Structural Details
 Cyanovirin-N is an 11 kDa protein (100 residues)
 Used 15N and 13C labeling to get the structure
 Used D2O exchange to get some assignments
 Protein is symmetric-has a 2-fold axis
 The sample used was 1.4 mM
 For total restraints per residue:
 2509 restraints/100 residues = 25 restraints/residue
NOEs per Residue
A general rule for resolution of NMR structures (to
keep nomenclature consistent with Crystallography:
Less than 5 NOEs per residue = 8 –10 A structure
6-12 NOEs per residue = 5 A structure
12-15 NOEs per residue = 3 A structure
16-20 NOEs per residue = 2.3-2.5 A structure
Over 21 NOEs per residue = 2.0 A structure
Clore GM and AM Groenborn (1991) Science
NMR structure of cyanovirin-N
This is a collection of over 40 structures generated by “simulated
annealing”.
Secondary Structure
Final 30
structures
Parameters
Mean
structure
Figure 6.
fig6
Red = negative
charge
Blue = positive
charge
Yellow=hydrophobic
Comparison of the NMR and Crystal
Structures
NMR = monomer, X-Ray = domain-swapped dimer
In this paper, they reconcile the x-ray structure and NMR
structure by generating new ones.
They picked conditions to favor dimer formation in NMR
(refolding the protein in high concentrations or incubation of the
monomer at high concentrations and higher temperature).
In the crystal structure, they got a slightly different form, but it
(in general) the same as the previous crystal structure.
Used the crystal structure as the initial model for solving the
solution structure of the dimer.
Figure 1
NMR Parameters
Looked at the resonance corresponding to Trp49 Ne1H (in
b strand 5). This residue serves as part of the interface
between the 2 domains- in both the monomer form (A and B
– from the monomer) and the domain-swapped dimer form.
Used a suspension of Pf1 to help align the protein. Why?
R1 = K g2I g2S rIS-6 tc,
Figure 7
How Pf1 aligns proteins, DNA (and
RNA?)
Bo
Figure 2
Figure 3
Figure 4
Figure 6
Homework Handout
Question 1.
Nucleus
Spin Number
Natural
Abundance, %
1
1-0 odd 1/2
99.98
2
2-1 even, odd 1
0.015
12
12-6 even, even 0
98.9
13
13-6, odd, even 1/2
1.1
14
14-7, even, odd 1
99.62
15
15-7, odd, odd 1/2
0.4
16
16-8, even, even 0
99.76
17
17-8, odd, even 5/2
0.039
19
19-9, odd, odd 1/2
100
23
23-11, odd, odd 3/2
100
31
31-15, odd, odd 1/2
100
59
59-27, odd, odd 7/2
100
H
H
C
C
N
N
O
O
F
Na
P
Co
Question 2A
A.
NMR
X-ray
Solvent conditions more physiological Solvents- uses a lot of
detergents, salts
Nondestructive
Destructive
Sees protons, connectivities first,
Sees overall physical structure
then model physical structure
first, have to model in protons,
connectivities
Can take months, sometimes years
Takes minimum 1 month, up to
a year
Can use as little as 0.1 mM to 1 mM
Needs several mM of protein to
screen for crystals
Can be used to study dynamics
Motion is bad
Question 2B
B. Because of their differences, they can be used to compliment one another,
however it has to be certain that the solvent conditions did not change the overall
structure of the molecule. Small changes can be accommodated, but large ones
will cause contradictions.
1. NMR can confirm that crystallization doesn’t give a structure due to solvent
conditions.
2. NMR can help solve the phase problem for crystallography or crystallography
can start as the initial model for NMR, but 1 has to be true first.
3. If a region appears to be mobile in the crystal, NMR can confirm this and get a
minimized structure of the mobile region to complete the picture.
4. If working together, a relatively large protein (25-40 kD) can be solved fast.
Question 3
In the NOESY below, the off-diagonal circles are crosspeaks that were in
the corresponding COSY, while the crosses are new crosspeaks in the
NOESY. Explain, using proton numbers, what each crosspeak tell us about
the molecule.
Things to remember:
Diagonal are crosspeaks to themselves
Peaks that are in the COSY, but not NOESY are
correlated to each other
New Peaks in the NOESY tell us about non-coupled,
spins that are close in space.
Question 3
Figure 8