Transcript 1D 1H NMR spectra of proteins

Resonance assignment in proteins
•in order to be able to actually solve the structure of a protein, we
first have to assign the spectrum. Each peak corresponds to some
1H nucleus within some amino acid residue.
•The sharp peak at -0.8 ppm is almost certainly a methyl.
Is it a valine, leucine or isoleucine methyl?
•suppose we knew it was a valine methyl. We still don’t know:
Which valine in the protein does it belong to?
•suppose we knew it was Val30. We still don’t know
Which of the two methyls of Val30 is it?
sequence of lysozyme:
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA
KFESNFNTQATNRNTDGSTDYGILQINSRWWCN
DGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVS
DGNGMNAWVAWRNRCKGTDVQAWIRGCRL
Levels of resonance assignment
• residue or “spin system” assignment:
is it a Val, Ile or Leu methyl?
• sequence-specific assignment:
is it in Val 30 or Val 87?
• stereospecific assignment:
is it the pro-R or pro-S methyl of Val 87?
Part I: 1H spin system assignment
•
•
a spin system is a set of 1H resonances connected (either directly or in
a “relayed” fashion) by 1H-1H scalar couplings (also called J couplings)
generally this means networks of 1H in which each 1H is connected to
some other member of the network by three or fewer covalent bonds-The reason for defining it this way is that four and five bond couplings
are generally too small (<1 Hz) to be seen in NMR experiments.
Hc
“relayed”
connection
H
H
geminal coupling
(two-bond)
J ~ -12 to -15 Hz
H
H
vicinal coupling
(three-bond)
J ~ 2-14 Hz
Ha
Hb
example of a
spin system
most residues in proteins have one 1H
spin system
3-bond couplings
2-bond couplings
spin systems never span more than a
single residue--the carbonyl group assures
that the closest coupling between residues
is never fewer than four bonds...
CO2H
C
H
H
CH3
C
H
H
H
4-bonds,
too far!
C
C
N
C
N
C
H
O
H
O
Glu
Ala
H
3-bond couplings
2-bond couplings
C
H
H
C
C
C
C
H
C
H
C
H
H
H
C
N
C
H
O
Phe
 Some residue types contain
multiple spin systems
 for example, phenylalanine
has a separate spin system for
the aromatic ring and for the
amide-alpha-beta linkage
Homonuclear J-coupled 2D spectra
HB
scalar or J-coupled protons
HA
HB
diagonal peak:
correlation
of a resonance with itself
crosspeak:
correlation of two
different resonances
by through-bond
1H
HA
HB
chemical shift (ppm)
HA
scalar coupling connection
1H
chemical shift (ppm)
COSY and TOCSY spectra
COSY is a type of 1H-1H homonuclear 2D spectrum that shows crosspeaks
between 1H nuclei with geminal or vicinal coupling (two or three bond)
TOCSY is similar but includes “relays” of coupling so that crosspeaks can
be observed between 1H nuclei connected by more than three covalent
bonds.
direct two and three
bond seen in COSY
“relayed” connections
seen in TOCSY
H
H
geminal coupling
(two-bond)
J ~ -12 to -15 Hz
H
H
vicinal coupling
(three-bond)
J ~ 2-14 Hz
Ha
Hb
Hc
2D COSY and TOCSY-->spin systems
•
COSY and TOCSY can be used to
assign spin systems through
recognition of coupling patterns
•
important to note that recognition of
the patterns at right also takes into
account qualitative chemical shift
information--for example, Gly and
Ala are distinguished from each
other by the fact that the coupled
resonances of Gly are both in the
alpha region (3-5.5), while for Ala
we see a coupling between
resonances in the alpha region and
in the methyl region (0-1.7). So the
Ala crosspeak will be much further
from the diagonal (see figure).
o crosspeaks visible in COSY
+, * crosspeaks visible in TOCSY
Example of lysine spin system
CO
HN
a
b
g
d
e
NH3+
Ha He
Hb
Hd
Hg
Hg
Hd
Hb
He
Ha