Biomolecular NMR Spectroscopy: An Introduction

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Biomolecular NMR Spectroscopy An
Introduction Chuck Sanders Dept. of
Biochemistry Rm 5110, MRB III (936-3756,
chuck.sanders_at_vanderbilt.edu)
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http//drx.ch.huji.ac.il/nmr/whatisnmr/basisnmr.jp
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www.chem.ucalgary.ca/courses/ 351/Carey/Ch13/1301.
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NMR Resonance Frequencies are in the 10-1000 MHz
Frequency Range Radio Wavelengths Recall E
hn NMR is a low energy/low sensitivity
technique
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www.york.ac.uk/depts/chem/services/
nmr/pictures/nmrtube.gif
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Primary Biomolecular NMR Isotopes 1H (most
sensitive, no labeling required) 13C (usually
requires biosynthetic labeling) 31P (no
labeling required) 15N (requires biosynthetic
labeling) 2H (requires biosynthetic
labeling not usually observed, but used to
get rid of 1Hs) Each isotope has its own
intrinsic Larmor resonance frequency. For
these nuclei, labeling is merely isotopic
enrichment- no potentially perturbative probe is
required.
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In real molecules, resonance frequency is not
exactly equal to the nulceus Larmor frequency.
Instead, the local magnetic field generated by
the electrons In the surrounding atoms/bonds
tweaks the frequency just a bit. The
observed tweaking factor is called the
CHEMICAL SHIFT and is why different protons In
a protein give rise to different peaks. The
existence of the chemical shift is key to the
usefulness of NMR.
Some implications Each resonance in a
biomolecule has its own chemical shift. When
structure or environment changes, shifts change.
Local magnetic field generated by Electrons in an
aromatic ring.
www.chemistry.ccsu.edu/.../teaching/
472/nmr/factors.html
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1H NMR Spectrum of Lysozyme
From Varian Instruments, 900 MHz
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Multidimensional NMR A tool for resolving peaks,
for assigning spectrum, etc. Here is a 2-D
1H/15N spectrum. Each dot represents a directly
bonded amide1H-15N pair crosspeak resonance.
1-D 1H spectrum
1-D 15N spectrum
stingray.bio.cmu.edu/ web/nmr/hetero/
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Assigned 1H/15N Spectrum of Diacylglycerol
Kinase (80 of assignments completed).
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Things Which Can Be Learned Using NMR By Simply
Following Changes in Positions of Assigned
Peaks Affinity of ligand binding. Affinity of
protein-protein or protein-nucleic acid
binding. Identity of residues in binding sites
of protein or RNA/DNA. Monitor protein folding
and unfolding as function of pH, temperature, or
denaturant. Can identify if some domains are
more stable than others. Assigned Chemical
Shifts Also Provide a Map of the Secondary
Structural Elements in a Protein.
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The Shutter Speed of NMR If a nucleus spends
time in two or more environments (exchanges),
does one see multiple peaks (1 for each
environment) or just a single average peak? The
answer If the exchange rate is rapid compared
to the difference in resonance frequencies for
that species in each environment, then only an
average peak is observed. (Fast exchange
conditions). If the exchange rate is slow
compared to the differences in resonance
frequencies, then peaks arising from each
environment can be observed. (Slow exchange
conditions). NMR can be used to measure rates if
intermediate exchange conditions apply, where
partial averaging is observed.
Slow or no exchange
The above case is for a single peak from a
molecule which populates two different
environments with equal populations.
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The Social Life of NMR Spins Interactions
Between NMR-Active Nuclei J coupling nuclei
talk through the mediation of electrons
connecting the nuclei through covalent bonds.
J couplings give dihedral angles
conformational information! Dipole-Dipole
Cross-Relaxation the Nuclear Overhauser Effect
(NOE) Nuclei which are close in space
influence each others relaxation behavior,
measured as NOEs. NOEs give distances between
nuclei this is the primary means by which
protein structure is determined by NMR. (NOEs
are measured as crosspeak intensities in a 2- or
3-D spectra). Residual Dipolar Couplings A
newly-accessed form of structural data. This
coupling results from direct (through space)
dipole-dipole magnetic interactions between
proximal nuclei. These couplings give
orientation of the vector between the interacting
nuclei with respect to a fixed reference axis.
This information can be used to solve protein
structure on its own, but most often is combined
with NOE and J coupling data, leading to very
accurate and precise structures NMR Relaxation
Rates These are strongly coupled to molecular
motion. NMR is THE technique for studying
molecular conformational motions.
http//drx.ch.huji.ac.il/nmr/whatisnmr/coupling.jp
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Example of how orientational data such as that
provided by residual dipolar couplings can lead
to 3-D structure.
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Of the ca. 18,000 biopolymer structures currently
deposited at the Protein Databank, about 2900 are
NMR-derived.
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Summary of Biomolecular NMR Applications NMR
can serve as reporter method to monitor ligand
binding, biomolecular interactions,
folding/unfolding. Can be used to measure
binding/dissociation and conformational exchange
rates. Can be used to determine secondary,
tertiary, and quaternary structure at high
resolution. Can be use to identify location of
binding sites. Can be used to study
motions. Can be used to probe protein folding
pathways. Can be used to monitor biomolecular
reactions.
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NMR Practical Aspects and Limitations Sample
Volume about 0.6 ml Required concentration
historically about 1 mM (20 mg/ml for a 20 kD
protein). This is a weakness of NMR it requires
a lot of sample. HOWEVER, there is a new
generation of NMR signal detection technology
(cryoprobes) which enhance the sensitivity of
NMR by a factor of 5-10. Thus, submillimolar
concentrations are now feasible. NMR sensitivity
is roughly proportional to the magnetic field
strength to the 3/2 power, and so the development
of very high field NMR magnets has also helped a
lot to lower sample requirements. Expense NMR
spectrometers used to study biomolecules run from
about 0.5 million to 5 million US the magnet is
the most expensive component (bigger is better is
more pricy!). Instrument time typically costs
5-20 per hour. Experiments may require anywhere
from half an hour (1-D experiments) to several
weeks (complete set of multidimensional
experiments to solve 3-D structure).
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Molecular Weight Limitations NMR spectra can be
acquired even of solids. Indeed, solid state NMR
is an extremely active and fertile field.
Moreover, 1-D solution NMR works fine even for
very large molecules and complexes. However, the
multidimensional NMR experiments normally used to
assign spectra and to solve protein structures
tend to stop working when the effective molecular
weight reaches about 40 kDa (where resonance line
widths approach the size of the NH coupling
constants which are critical to the success of
most multidimensional NMR experiments). The
vast majority of protein structures solved by NMR
are of proteins which are smaller than 25 kDa.
However, this traditional limitation of NMR is
being broken down due to the TROSY effect and
related NMR experiments. Determining the
structures of molecules and complexes in the 100
kDa range is now clearly feasible.
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JNH
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Diacylglycerol Kinase a 40 kDa Membrane Protein
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800 MHz Spectra of DAGK in Micelles (100 kDa)
15N
1H
Traditional HSQC
TROSY
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Assigned 1H/15N TROSY Spectrum of Diacylglycerol
Kinase (80 of assignments completed).
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Three Examples of Applications of NMR to Solve
Biological Problems
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Diacylglycerol Kinase a 40 kDa Membrane Protein
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Biochemistry 40, 5111 (2001)
100 Active
70 Active
TROSY (1H-15N) spectra of two samples of the same
diacylglycerol kinase mutant. 1 sample has a 30
misfolded population. NMR show that the
misfolded form has a specific, well-defined
conformation.
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Adenylate Kinase ATP AMP 2ADP
What parts of ATP supply the most binding
energy? How mobile are those parts after binding?
Biochemistry 28, 9028-9043 (1989)
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1H NMR peaks of AK were monitored as the enzyme
was titrated with ATP and various isolated
parts of this nucleotide. By monitoring peak
shifts as a function of ligand concentration,
binding constants were determined.
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Most of the binding energy driving ATP binding to
AK comes from the triphosphate moiety of ATP.
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The mobilities of the AK-bound adenine and
triphosphate moieties were determined by
measuring 2H NMR relaxation times (T1 and T2) and
derived correlation times (the smaller the more
mobile). For these studies a deuterated ATP
analog was employed.
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The triphosphate group of ATP provides most of
the binding energy, but retains considerable
mobility at the active site of AK. The adenine
moiety is rigidly bound at the active site, but
provides little binding energy.
Samson and Prometheus both were tightly bound,
but had very different local mobilities in each
case with tragic consequences.
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Dorothee Kern, Brian Volkman, David Wemmer et al.
Nature 401, 894-898 (1999) Science 291,
2429-2433 (2001) When a signal transduction
protein gets activated by being phosphorylated,
which of the following applies? Inactive
Conformation Active Conformation OR. Is there
a preexisting equilibrium between active/inactive
states which can be shifted? Inactive
Conformation Active Conformation Upon
phosphorylation, equilibrium shifts to favor
active form Inactive Conformation Active
Conformation
Phosphorylation acts as switch
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Kern et. al., Nature
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Kern et. al., Nature
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Kern et. al., Science
P-NtrC Mobility map
NtrC Mobility map
Blue NtrC Gold P-NtrC
Map of spatial differences between NtrC and P-NtrC
Map of chemical shift differences between NtrC
and P-NtrC
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Based on these results it is clear that
phosphorylation shifts a pre-existing equilibrium
rather than acting a switch to trigger a new
conformation.
Kern et. al, Science
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SHOW THE MOVIE NOW
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If you would like to see Vanderbilts High Field
NMR Center go with Chuck now.