NMR Nuclear Magnetic Resonance

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• NMR- Nuclear Magnetic Resonance
• Gives dynamic information on a protein that a
  crystal doesnt
• Certain nuclei possess a property known as spin
• Spin refers to the nuclear spin angular momentum
  which is purely quantum mechanical property that
  has no classical analog. Its represented by I
  spin quantum number
• 3 classes of nuclei
• Nuclei with odd mass number have ½ integral spin
• Nuclei with even mass numbers and an even charge
  have no spin. 12C, NMR inactive
• Nuclei with even mass numbers and odd charge have
  integral spin.
• 14N, broad lines
• Category 1 is the most used in NMR because I ½
  and can use 1H, 13C, 15N

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What does I ½ mean? It means that such a
nucleus can orient itself in 2I 1 ways when
placed in a magnetic field 2I 1, I ½
2 2 nuclear orientations so 2 possible nuclear
energy states If we represent these orientations
as vectors what we can say is that in the absence
of any external force or field, these 2
orientations are identical and have the same
energy you cant tell them apart. But what
happens if you introduce an external magnetic
field? Of size Bo?
Energy states separate out with introduction of
external magnetic field
ß
E
? E
a
No field
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The energy difference ? E h ? (normal
spectroscopy) -can flip between states at
different energy levels We can see that the
stronger Bo (ext field) then the further apart
these energy levels so ?E a Bo. The larger the
field the larger the separation. h
plancks constant ?E h ? Bo ? some
constant Bo magnetic field ?
magnetogyric ratio tells you have receptive a
nuclear type is compared to another So
essentially if we could cause a transition
between the state, this would be some sort of
spectroscopy The frequency required to induce
such transitions is in the MHz range
radio-frequency referred to as RF From a basic
standpoint if you put in RF at the appropriate
MHz frequency then you will induce such a
transition This is the basic unit of NMR
phenomenon!
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?H 4 ?C ?H -10 ?N
Sensitivity also depends on natural abundance 1H
vs. 13C 1001 So you would imagine H vs. 13C
is 400 times better at natural abundance and 10
times better than 15N
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It turns out for a variety of complex reasons
that NMR is not like other types of spectroscopy
(ex. UV) If you fire in a photon with the right
RF frequency to cause a transition from
ß a then this states
lifetime is so short that we cant see it The
upshot of this is that we cant get an NMR signal
from just one nucleus. We need BILLIONS
though. We measure coherence.
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The billions of nuclei all act together when we
put in a RF pulse Setting up what is essentially
a COHERENT effect between the a and ß states.
ß Billions this coherence (like
constructive interference) a -oscillates
at the frequency between the 2 levels and
last for seconds So we put a billion nuclei
into a magnetic field, separate the nuclear
energy states and cause a coherence to be set up
that we can detect at the specific frequency
between those 2 levels. Any problem? YES
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The levels are very close in energy 1 in 105
nuclei prefer to go in a rather than ß ß
?E a The distribution of population between
the a and ß states is given by the Boltzmann
equation. ?E a Bo Therefore, more go
into a state as the external field gets bigger.
Makes experiment more sensitive. But its still
small ? very sensitive technique
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Make our protein express, purify, search for
conditions Then put it in a magnet. Lets
just think about 1H nuclei for now. Magnet
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Place current in coil and then cool. The
electrons move forever because made a super
conductive wire by keeping temperature at 4
Kelvin. There is no resistance in the wire.
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So, RF gets delivered and hits the nuclei
coherence is set up and detected If there was
only 1 nuclear type then 1 signal at one
frequency easy to see H1-outside H2-hydrop
hobic core H3-cavity 3 types different
electronic environments The different electronic
environments shield them to different extents
from the main Bo field.
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So, if they have different Bo responses then they
have different ?Es and so different
frequencies 3 different frequencies from 3
different environments All superimposed not
easy to see
10 Hz
30 Hz
20 Hz
H1 10Hz H2 20 Hz H3 30 Hz
All superimposed not easy to see More shielded
proton the lower the frequency
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Take the oscillating waves Fourier transform
them get their frequencies
30 20 10 Chemical Shift
Differences So each 1H in a different
environment has a different chemical shift
property a nucleus possesses which is dependent
on its electron environment. More electrons the
more shielded from main field so will have
different chemical shift then a lesser shielded
electron. Normally measured in PPM We
also notice some fine structure here. Whats that?
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Electrons from HA ? HB and vice versa so HA knows
about HB and knows if its in its a or ß state
can be either so HA sees HaB and HßB Produces
2 lines at HA frequency Similarly HB sees HaA
and HßA ? 2 lines at HB The two lines split by
same amount and next to each in protein structure
J J HA
HB
J Scalar Coupling in Hz Why lines are split
Thru bond interactions
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Example of coupling in a chemical shift
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Scalar Coupling is through bond interaction So
if we see scalar coupling we know that 2 nuclei
are next to each other through bonds. Chemical
shift structure info different positions on a
molecule Scalar coupling structure info
through bond connections There is one other type
of interaction that is very, very
important Not close in bonds (no
HA HB scalar coupling) But close in
space lt 5A They can sense each others
presence as the molecule tumbles. Their dipoles
are coupled Dipolar coupling through space
interaction gt 5 Angstroms apart
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This thru space dipolar coupling interaction
produces an effect known as the Nuclear
Overhauser Enhancement (NOE) - used to
accurately determine distance Which allows us to
very accurately monitor and calculate thru space
distances.
H
NOE _at_ a fixed distance. Use this as a reference
H
More often we classify them as Weak Medium
Strong 5A 4 - 3/2 A 1 - 2 A
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J / NOE
H H C C H C C H
Helix (cis) J small close in space and
attached by bonds NOE
Use NOE and J to determine structural position of
protein
Sheet (trans) J big distance gt5A Attached by
bonds NO NOE
J / No NOE
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So we can see different environments from
chemical shifts, adjacent thru bond connections
from scalar couplings and thru space interactions
from dipolar couplings/ NOE Depending on
the structure look _at_ all these possible thru
space connections
For a helix
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What about helices next to each other? What
about ß-strands / sheets?
i ? i 4 / 3
interhelix
NOE between N-H and N-H across strand ? dNN No
NOE between adjacent residues because too
far NOE between adjacent reidues H-H ? da
No daNs on same residue No sequential dNNs if
you see, there are interstrand
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And turns.
Adjacent residues
Cross-strand
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So we can connect things accurately thru space to
define different strands, sheets, helices and
turns and their 3D position with respect to each
other. Thru BOND scalar coupling connections are
used to identify different amino acids. Each has
a pretty unique coupling network. Will
generate clearly different patterns So 1.
identify amino acids using chemical shifts and
scalar coupling patterns 2. (i)
sequentially and (ii) 3 dimensionally arrange
those amino acids by using NOEs This is called
(i) sequential assignment (ii) structure
generation
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Sequential NOEs will have unique scalar coupling
1 amino acid
Peptide backbone
No J between neighboring amino acids because no
through bond protons present
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So if you say link 3 together you can find that
run in the primary sequence and assign each
resonance / peak to a specific proton in the
protein. Ex. Amide of ALA32 ßH of Leu 86
etc. If you have every peak assigned, then you
can connect them all via NOEs 3D
structure Scalar coupling use to ID the amino
acids NOEs use for sequential
ordering Problem The bigger the proteinthe
more protons -OVERLAP
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These experiments can also be made to disperse
the information into 2 dimensions.
Movement of frequencies goes both ways
HB HA
HA HB
HA HB C C
Establishes connectivities through bonds 2D
Correlated Spectroscopy COSY experiment Connect
s scalar coupled, through bond connected nuclei
in 2 frequency dimensions.
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We have a lot of overlap in 1D spectra - only
one dimension to disperse info into
HA HB C C
Wouldnt it be great if we could transfer the
magnetization of one nucleus, HA to HB (and vice
verse)? So that in a spectrum we would have a
peak HA and a peak HB But also a peak HA
-gt HB a peak HB -gt HA Use the scalar
coupling to transfer magnetization can we do
this? Yes we can there are NMR experiments
that can do this
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HB HA
HA HB
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2 dimensions gives you more resolution Each
cross peak represents a connection thru bonds
2D Cosy Cant jump over C-O bond (must have
neighboring amino acids) because no protons to go
thru
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Examples of COSY patterns for amino acids. The
strong resonances of the diagonal (large circles)
give chemical shifts of indicated H atoms. Cross
peaks give thru bond interactions.
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Can you do this with dipolar thru space
couplings? Send magnetization from one proton to
another thru space? 2D NOESY Each cross peak
represents a thru space connections from one
proton to another (lt 5A) Amount of peaks in line
tells amount of stuff in a shell around a
proton Closer to the stronger ? 1A Furthere
apart the weaker ? 5A
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We talked about only protons In proteins 12C
is dominant (99) and 14N too (approx
99.9) Both these are INACTIVE NMR WISE
useless 13C and 15N have I ½ but have very low
natural abundance can we do anything about
that? Yes when you grow your proteins
introduce 15N via ammonium sulphate/chloride via
glucose In growth medium -- 100 incorporation
of 13C and/or 15N
Isotopically labeled and now NMR active except
O We can pass magnetization anywhere via scalar
coupling
O H H O
H --13C-------13C--------15N--------13C--------1
3C------15N 13R
13R
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Methyl
ß
a
So in a C- H group we can pass magnetization H ?
C Looks fabulous but bigger proteins even make
there 2D spectra look messy and overcrowded with
peaks because more nuclei present 1D ? 2D 2D ?
3D?
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A peak belongs to what? The amide proton shifts
about the same. But what if Nitrogens had
different shifts? Could pull it apart.
Step 3. Pull apart
Step 2. Proton N chemical shift
Step 1. NOSY Grey, black, and white dots 3
separate residue peaks
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Thru Bond
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Thru space
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a C N H
3D HNCA 1 peak that connect every NH and a C group
Eventual goal using all these 3D experiments is
to Assign every peak in every spectrum to a
specific atom in the protein Know which peak is
which proton 1H ex. ß- proton of Ser 38
NH proton of Leu 94 etc. The use NOEs between
pairs, to determine distance input this data in
a program 3D Structure