Transcript nOe

Areas of Spectrum
Examples of aliphatic region correlations
1.52
Ala
3.95
Thr
Asp
The fingerprint region – the 2nd region of interest in the COSY spectrum
Areas of Spectrum
COSY Fingerprint region correlating NH-aH protons
COSY Spectrum of a small protein
Aliphatic
Fingerprint region
Total correlation spectroscopy - TOCSY
90o
t1
t2
Water Presaturation
Spin locking field
The spin locking field (a series of rapid 90o pulses of
varying phase) effectively averages the coupling 1H-1H
coupling constants over the entire spin system.
The dispersion of the NH-aH region allows correlations along
the entire system to become visible.
Homonuclear Hartmann-Hahn or TOCSY experiments
Under these conditions magnetisation is transferred very efficiently,
at a rate determined by J, between coupled nuclei. The longer the
mixing time, the further through the spin system the magnetisation
propagates.
J13=0.2 Hz
1
2
3
J12=7 Hz
J23=5 Hz
Even if J13 is very small, will still see transfer to it via 2
8.83ppm
3.95ppm
1.52 bCH3
1.52ppm
ALA 49
Ala49 3.95 aH
1.52 bCH3
3.95 aH
8.83ppm
Connecting spin systems – The nuclear Overhauser effect (nOe)
At this point, we have used COSY and TOCSY to connect spin systems. i.e. if there are 8
arginines in the protein, we would aim to find 8 arginine patterns. Overlap or missing
signals may hamper us in this initial goal. The next step is to use NOESY experiments to
sequentially link the amino acid spin systems together.
The nuclear Overhauser enhancement provides data on internuclear distances. These
can be more directly correlated with molecular structure.
Consider 2 protons, I and S, not J-coupled but close in space
W1 is the normal transition probability that gives rise to a peak in the spectrum
bb
W1s
W2 flip flip
W1I
W0 flip flop
ba
W1I
ab
W1S
aa
W1 requires frequencies or magnetic field fluctuations near the
Larmor precession frequency i.e. (e.g. 500 MHz at 11.1 Tesla).
W2 requires frequencies at wI + ws, or to a good approximation, 2wI or 109 Hz
Wo is a zero quantum transition that requires frequencies at wI-ws, i.e. just the
chemical shift difference of the protons which could be 0 to a few 1000 Hz)
In the energy level diagram for a 2 spin system, it is the transitions that involve
a simultaneous flip of both spins (cross - relaxation) that cause NOE
enhancements.
A transition corresponding to a given frequency is promoted by molecular motion
at the same frequency. Small molecules in non-viscous solvents tumble at rates
around 1011 Hz, while larger molecules such as proteins tumble at rates around
107 Hz. For small molecules, W2 will be greater than W0 and this is the dominant
mechanism for producing NOE enhancements (which turn out be positive)
For larger molecules W0 will become greater than W2 and this becomes the dominant
mechanism leading to NOE enhancements (that are now negative).
Rotational correlation time tc
rotational correlation time [in ns] is approx. equal to 0.5  molecular mass [in kDa]
For a small molecule, tc is small (~0.3ns) and the product wtc is << 1. In this extreme
narrowing limit, rotational motions include 2wo (i.e. fast motions) and W2 is preferred.
In large molecules (PROTEINS!), the tumbling is slow and wtc > 1. Wo connects
energy levels of similar energy so only low frequencies are required. Therefore this is
the preferred mechanism in large molecules. It is known as cross-relaxation.
In the 2D NOESY experiment, an additional mixing time is added to the basic
COSY sequence. The result is a build up of magnetisation from one nucleus to a
close neighbour.
90o
t1
90o
90o
Mixing time
t2
Presat
(magnetisation components of interest lie along –z). Cross relaxation now occurs to
nearby nuclei.
The NOE operates ‘through space’, it does not require the nuclei
to be chemically bonded. The build-up is proportional to the
separation of the two nuclei
1
NOE µ 6
r
nuclear separation
If we calibrate this function by measuring a known distance in the
protein and the intensity of the NOE, we can write
1
NOE = k 6
r
where k is a
proportionality
constant
The power of the NOESY experiment is that the intensity of an
NOE peak will be related to the nuclear separation.
Strong NOE crosspeaks - 2.5 Å
Weak NOE crosspeaks - 2.5-3.5 Å
Extending the mixing time will permit nuclei separated by 5Å - not
all spin systems will give a detectable peak though. So the absence of
a peak does not preclude close approach. Similarly a weaker
crosspeak does not always prove a larger internuclear distance.
Therefore tend to be cautious and define distance ranges.
Strong (1.8-2.5Å), medium (1.8-3.5Å), weak (1.8-5.0Å).
Since this works through space we can use the NOE to connect spin
systems that we assigned with the COSY and TOCSY spectra.
1
2
Sequential ‘walking’ with sequential nOes
Fingerprint region
dH
of a 2D NOESY
TOCSY
gH
bH
3
4
5
COSY
9.0
COSY
TOCSY
NOE
8.0
O
C
CH3
N
C
C
H
H
O
NOE
COO
CH2
H
H
O
N
C
C
CH2
Ala
CH
H3C
-
CH3
N
H
H
7.0
NH
NH-NH Contacts
NOE
O
C
CH3
N
C
C
H
H
O
1
COO
H
H
O
N
C
C
CH2
Ala
CH
H3C
dH
-
CH2
N
H
gH
2
bH
3
H
4
CH3
5
9.0
The ‘NH-NH’ region provides an
additional source of sequential
contacts - note the symmetry
around the diagonal and that
this contact does not give direction.
8.0
7.0
aHi-NHi+3
aHi-NHi+1
i+3
H
i+2
An a-helix can be recognised
by repeating patterns of short
range nOes. A short range nOe
is defined as a contact between
residues less than five apart in
N
i+4 the sequence (sequential nOes
H
connect neighbouring residues)
NOE
H
For an a-helix we see aHi-NHi+3
and aHi-NHi+4 nOes.
i
A b-strand is distinguished by strong CaHi-NHi+1contacts and long range nOes connecting the
strands.
A long range nOe connects residues more than 5 residues apart in the chain.
Assignment of secondary structural segments
• sequential stretches of residues with consistent secondary
structure characteristics provide a reliable indication of the
location of these structural segments