Transcript Document
The number of protons yielding correlations in a 2D NOESY spectrum quickly overwhelms the space available on
A 2D map. 15N labeling can help simplify the fingerprint region but not the aliphatic region
These methods take advantage of large 1J coupling constants
2
H
C
H
JNC
1
1
C
N
1
H
JNH
1
H
JNC
H
1
JC'C
C
JNC'
C'
N
C
O
H
H
J HC
Coupling
Magnitude (Hz)
1
93
1
7 - 11
2
4-9
1
15
1
55
1
140
JNH
JNC
JNC
JNC’
JCC’
JHC
H
Backbone assignment via 1J couplings
N
HNCA
C
H
H
H
i
i-1
N
2
H
C
C
C
i
C
N
1
H
O
H
3
H
2
H
Start here – excite
protons with a
proton 90o pulse
HN(CO)CA
H
C
H
H
2
1
C
N
C
C
N
i
i-1
i-1
H
C
O
H
H
H
3
H
Slice from HNCA (at the 15N shift
of I44, T14, R74..). Each pair of
peaks correlates a C(i) and C(i-1)
with the 1H and 15N shift of
residue i.
Slice from HN(CO)CA (at the
15N shift of I44, T14, R74..).
Each pair of peaks correlates the
C(i-1) with the 1H and 15N
shift of residue i.
Stage 2. Sidechain assignments completed with HCCH-COSY and
HCCH-TOCSY for example.
The HCCH experiments provide connectivities of the aliphatic side
chains of individual amino acid residues.
Complete assignments can be obtained if the backbone
assignments and the side-chain assignments can be connected via
the 13C shifts.
An example. 13C shifts of Isoleucine
5-15ppm
13
CH3
1 25-30ppm
CH2
H
30-35ppm
2 15-20ppm
13
15
N
H
13
13
CH3
13
13
C
C
C
H
50-65ppm
O
We know the 13C shifts
from the backbone
assignment
Attempt to gain complete 1H, 15N and 13C chemical shift
assignments. We can now resolve uncertainty in NOEs
we observe.
CH3 0.82ppm
CH3 of
Ile 2
1
H 0.55 ppm
3 ngstroms
CH3 1H 0.55ppm
CH3 of
Ile 1
H3C
8 Angstroms
CH3
0.82 ppm
These 4 methyls would give an ambiguous network of
possible NOEs. But suppose we knew that the 13C shift of
the CH3 of Ile 1 was 9.3ppm and the CH3 of Ile 2 was 13
ppm.
Far larger proteins can now be tackled…44kDa
Simian immuodeficiency
virus (SIV) ectodomain
used to fuse with host
white blood cells
Types of Spin Relaxation
•Longitudinal or spin-lattice relaxation (T1 )
- recovery of longitudinal magnetization
- establishment of thermal equilibrium populations
- exchange of energy
•Transverse or spin-spin relaxation (T2 )
-decay of transverse magnetization
- no exchange of energy
- increase of entropy
T1. Build up of longitudinal
magnetization when field is
switched on
Mz (t) = Mzeq [1- exp{- (t-ton) / T1}]
Equilibrium longitudinal magnetization
Spin-lattice relaxation time OR
longitudinal relaxation time
Inversion of longitudinal magnetization by π pulse
180o rotation about x-axis
Recovery of longitudinal magnetization after π pulse
1
2
Rotational correlation time c
small molecules tumble more quickly
large molecules tumble more slowly
rotational correlation time [in ns] approx. equal to
0.5 molecular mass [in kDa]
1 kDa = 1000 atomic mass units
Simple theory of T1
2
T11 2 Bran
(
c
1 o c
2
Larmor frequency
spin-lattice relaxation rate
constant
rotational correlation time
mean square amplitude of
fluctuating fields
Precession of Transverse Magnetization
Bo
z
z
z
xy plane
y
y
x
x
Mx
y
x
Mx (t) = Mzeq sin(t) exp{-t / T2}
Time
decay time constant =
spin-spin relaxation time OR transverse relaxation time
My (t) = -Mzeq cos(t) exp{-t / T2}
My
oscillation at the Larmor frequency
The transverse magnetization components
oscillate and decay
Time
Transverse relaxation or T2
decay
transverse magnetization is excited by first
pulse along –y-axis
transverse magnetization dephases due to
field inhomogeneity during the interval
/2. “Black” vectors rotate faster than
“grey” vectors
Problems with higher molecular weights and how to
overcome them
1
v
T2
v
is the
line-width
in Hz
at half peak
height
Comparison of T1 and T2
rapid motion (small
molecule non-viscous
liquids), T1 and T2 are
equal
Slow motion (large
molecules, viscous
liquids): T2 is shorter
than T1.
Sensitivity of an NMR Experiment
S N N
3/2
exc det
S /N
N
exc
det
Bo
NS
T2
3
2
1
2
1
2
Bo NS T2
Signal to noise ratio
Number of spins
sample concentration
Gyromagnetic ratio of excited spins
isotope labeling
Gyromagnetic ratio of detected spins
out and back experiments
Static magnetic field strength
magnet “size”
Number of scans
measurement time
Transverse relaxation time
molecular weight
Rattle page 46 and 47