Transcript Homonuclear 2D Experiments

Today’s Lecture
13) Mon, Oct 30: Assignments: I
a. Important homonuclear (e.g. 1H) experiments
b. Small molecules
c. Peptide assignments
A. S. Edison
University of Florida
2006
Homonuclear 2D Experiments
Name
Type of correlation
Notes
COSY
Correlated
Spectroscopy
1H
atoms separated by 2 or 3 covalent
bonds and mediated by J-coupling
(sometimes up to 5 bonds). Several
variants available.
Only pairs of coupled
atoms (compare to
TOCSY). Cross peaks
antiphase.
TOCSY
Total Correlation
Spectroscopy
1H
atoms separated by 2 or 3 covalent
bonds and mediated by J-coupling. All 1H
in a network of coupled spins will be
correlated
Correlations between
networks of coupled spins.
Very useful for side-chains
in peptides/proteins. Cross
peaks in phase.
NOESY
Nuclear
Overhauser Effect
Spectroscopy
1H
atoms up to ~5 Å in space mediated by
dipolar interactions. The NOE is highly
dependent on molecular correlation time:
Small molecules have positive NOE and
large molecules are negative. Larger
molecules have faster transfer rates.
Intermediate sized molecules (~500 Da)
do not work with NOESY.
Crude but important
measure of atomic
distances; generally
categorize into “strong”
(<2.5 Å), “medium” (<4 Å),
“weak” (>4 Å). Spin
diffusion can cause
problems in accurate
measurements.
ROESY
Rotating Frame
Overhauser Effect
Spectroscopy
1H
ROESY uses a transverse
spin lock and is very similar
to TOCSY. For this reason,
ROESY experiments often
have more artifacts than
NOESY.
atoms up to ~5 Å in space mediated by
dipolar interactions. Unlike NOESY,
ROESY is the same sign regardless of
correlation time. This is especially useful
for intermediate MW where NOESY
doesn’t work.
A. S. Edison
University of Florida
2006
DQF-COSY: Double quantum filtered COSY to
clean up diagonal and reduce solvent
Dossey, A. T., Walse, S. S., Rocca, J. R., &
Edison, A. S. “Single Insect NMR: A New
Tool to Probe Chemical Biodiversity” ACS
Chemical Biology, 1 (8), 511–514 (2006).
A. S. Edison
University of Florida
2006
TOCSY: 60 ms DIPSI-2 mixing time
A. S. Edison
University of Florida
2006
ROESY: 400 ms cw mixing time
A. S. Edison
University of Florida
2006
TOCSY and COSY comparison
A. S. Edison
University of Florida
2006
ROESY and COSY comparison
A. S. Edison
University of Florida
2006
TOCSY OVERVIEW
•TOCSY (Total Correlation Spectroscopy) is capable of correlating all spins in a
coupled network (examples shown on the next slide).
•The “working end” of the TOCSY pulse sequence is an isotropic mixing sequence that
is a sequence of pulses that are designed to remove all chemical shift differences and
create a strong coupling environment. This is called the Hartmann-Hahn condition.
Sometimes the TOCSY experiment is called HOHAHA (Homonuclear HartmannHahn).
•Several mixing sequencings have been developed. The most popular are MLEV-17,
WALTZ-16, GARP, and DIPSI. These all apply a series of pulses with either different
phases or different lengths or both.
•The strong coupling product operator is similar to the regular (weak) coupling operator
but it includes IxSx and IySy terms.
•The net result of the TOCSY mixing sequence is the transfer of magnetization from I to
S along the same axis (e.g. Iz to Sz or Ix to Sx or Iy to Sy). The transfer depends on the
coupling constant but is fairly complicated because S can transfer to another spin, R,
and so on.
A. S. Edison
University of Florida
2006
TOCSY Pulse sequence
(the simplest of several variants)
90x
t1
Isotropic Mixing
MLEV-17: (90-y, 180x, 90-y, 90-y, 180x, 90-y)*n 60x
DIPSI-2: (320, 410, 290, 285, 30, 245, 375, 265, 370)*n
The numbers are pulse
lengths in degrees. The
red and black pulses are
180 degrees out of
phase. The element in
the parenthesis is
repeated n-times to get
the desired mixing time
(e.g. 20-80 ms)
A. S. Edison
University of Florida
2006
Spin Systems
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
CH3
Any set of protons in a chain of unbroken J-coupling interactions will
give rise to sets of TOCSY cross peaks. For example, each of the 3 sets
of peaks shown above will be correlated in a 2D TOCSY spectrum.
A. S. Edison
University of Florida
2006
TOCSY spectrum of a 16 amino acid peptide
1
Each amino acid will produce
a pattern of peaks that
represent the network of
coupled spins in that amino
3
acid. The highlighted spin
system shown in red is a
leucine. Note that there are
H1
two leucines with very similar
chemical shifts.
5
Zachariah, C., Cameron, A., Lindberg, I., Kao,
K. J., Beinfeld, M. C., and Edison, A. S.
“Structural Studies of a Neuropeptide
Precursor Protein with an RGD Proteolytic
Site” Biochemistry 40, 8790-8799 (2001).
7
9
7
5
H2
3
1
A. S. Edison
University of Florida
2006
NOE
•Transferring magnetization through scalar coupling is a “coherent” process.
This means that all of the spins are doing the same thing at the same time.
•Relaxation is an “incoherent” process, because it is caused by random
fluxuations that are not coordinated.
•The nuclear Overhauser effect (NOE) is in incoherent process in which two
nuclear spins “cross-relax”. Recall that a single spin can relax by T1
(longitudinal or spin-latice) or T2 (transverse or spin-spin) mechanisms.
Nuclear spins can also cross-relax through dipole-dipole interactions and other
mechanisms. This cross relaxation causes changes in one spin through
perturbations of the other spin.
•The NOE is dependent on many factors. The major factors are molecular
tumbling frequency and internuclear distance. The intensity of the NOE is
proportional to r-6 where r is the distance between the 2 spins.
A. S. Edison
University of Florida
2006
Enhancement
NOE vs. ROE
wtc
NOE goes through zero
NOE
Small peptides
~10 kDa
~33kDa
A. S. Edison
University of Florida
2006
NOESY pulse sequence
90f2
90f1
t1
90f3
t
frec
Like all 2D sequences, t1 is the variable time to collect frequency information in the indirect
dimension. The delay t is fixed and is the time during which the NOE builds up. You might
have guessed that chemical exchange can also happen during this time, and it is possible to
confuse an NOE peak with a chemical exchange peak, but techniques have been developed to
figure out which is which.
Notice that I have not indicated the phases of the pulses. The different f’s make up a phasecycle, and we can also adjust which axis from which to record the signal (frec). These are
designed to minimize artifacts and select for desired correlations. Most 2D pulse sequences
have phase cycles.
A. S. Edison
University of Florida
2006
NOESY and TOCSY spectra of a 16 amino acid peptide
Each cross peak in a
NOESY spectrum
indicates that the
nuclei resonating at
the 2 frequencies
are within 5 Å in
space.
1
H1
3
5
Notice the difference
between TOCSY
(black) and NOESY
(red).
7
9
7
5
3
1
H2
A. S. Edison
University of Florida
2006
1H-based
Peptide Resonance Assignments
H
CH3
Ca
N
C’
H
O
H
O
N
C’
Ca
CH H
CH3 CH3
N
H
TOCSY/COSY
NOESY/ROESY
A. S. Edison
University of Florida
2006
1H-based
Resonance Assignments
GFGDEMSMPGVLRFamide
F2
S7
G10
V11
3.9
L12
E5
R13
4.2
4.5
M6
G3
D4
F14
"NH"2
4.8
M8
8.7
8.4
ppm
8.1
7.8
A. S. Edison
University of Florida
2006
Next Lecture
14) Wed, Nov 1: Assignments: II
a. Important heteronuclear experiments
b. 3D NMR
c. Assignment strategies in proteins
d. Intro to protein structure determination
A. S. Edison
University of Florida
2006