Transcript spin-system assignments

Resonance Assignment for Proteins
Classical homonuclear (1H-1H) assignment
methods:
1. Spin system assignments
2. Sequence-specific assignments
3. Sequential vs. Main-chain Directed
Assignment
Modern methods: Use of heteronuclear shift
correlation, triple resonance experiments, etc.
Resonance assignments
•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 proton within some amino acid
residue. Is the sharp peak at -0.8 ppm a valine, leucine or
isoleucine methyl?
•even if we knew it was a valine methyl, which valine does it belong
to?
•even if we knew it was Val30, which of the two methyls is it?
sequence of lysozyme:
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA
KFESNFNTQATNRNTDGSTDYGILQINSRWWCN
DGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVS
DGNGMNAWVAWRNRCKGTDVQAWIRGCRL
Levels of resonance assignment
• spin system assignment: is it Val, Ile or Leu?
• sequence-specific assignment: is it Val 30 or Val
87?
• stereospecific assignment: is it the pro-R or pro-S
methyl of Val 87?
Classical protein NMR: the basic plan
In “classical” protein NMR, assignments are made by
using 2-dimensional experiments to establish
correlations between different 1H resonances.
Recognition of characteristic patterns and networks of
correlations then allows assignments to be made.
Resonances are correlated either “through-bond”,
mediated by the scalar coupling, or “through-space”,
mediated by the spin dipolar coupling (nuclear
Overhauser effect).
H
H
through-bond (J-coupling)
through-space (nOe)
Basic features of 2D spectra
HA
HB
2Å
chemical shift (ppm)
HA
HB
diagonal peak:
correlation
of a resonance with itself
1H
crosspeak:
correlation of two
different resonances
by short interatomic
distance or through-bond
connection
1H
chemical shift (ppm)
Spin systems and scalar coupling
networks
•
•
a spin system is a set of 1H resonances connected (either directly or
indirectly) by 1H-1H scalar couplings
generally this means networks of 1H in which each 1H is connected to
another member of the network by three or fewer covalent bonds-longer-range couplings are generally small, so experiments based on
resonance correlation via scalar coupling will generally not detect fourand five-bond couplings
Hc
indirect
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
2D COSY/TOCSY-->spin systems
•
•
•
•
•
COSY and TOCSY give crosspeaks
when resonances are linked
through scalar coupling
COSY gives crosspeaks when 2and 3-bond couplings are present
in TOCSY, longer range correlations
are seen due to relays of 3-bond
couplings
these two techniques can be used
to assign spin systems through
recognition of coupling patterns
recognition of the patterns at right
also takes into account qualitative
chemical shift information--the beta
methyl of alanine, for instance,
might be anywhere from ~0.9-1.7
but is never 3 or 4.
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
Hd
Hg
Hb
He
Ha
Sequence-specific assignments
•
•
•
•
•
suppose we have the sequence of our protein from some independent
measurement
suppose we’ve assigned an isoleucine spin system, and there’s only
one isoleucine in the sequence (unique), at position 48. Then we know
our isoleucine is Ile48.
there won’t be very many unique amino acid residues in a protein,
however.
but there will be many unique dipeptide sequences
but in order to use this fact, we need to be able to connect adjacent
residues.
unique residues (arrows)
and unique dipeptide
sequences in lac repressor
Linking spin systems using nOe’s
• because the nOe depends
upon interatomic distance and
not upon J coupling, it can be
used to connect spin systems
which are adjacent in space but
not part of the same spin system,
for instance two residues
adjacent in the sequence
•general nomenclature for
interatomic distance between
atoms A and B in residues i and j:
dAB(i,j)
• nOe correlations are denoted using the distance nomenclature,
e.g. “dbN(i,i+1) nOe” or “dbN (i,i+1) correlation”
• daN(i,i+1), dNN(i,i+1), and sometimes dbN(i,i+1) are used to
connect adjacent residues
2D NOESY: linking spin systems
4.HN/5.HN
5.HN/6.HN
diagonal: no
magnetization
transferred
crosspeaks: intersection
of chemical shifts of atoms
which are close in space,
i.e. < 5 Å
1H
6.HN/7.HN
3.HN/4.HN
1H
portion of 2D
NOESY of
P22 cro showing
dNN(i,i+1) correlations-can “walk” along the
chain from one
residue to the next.
Residues 3-7 shown.
Sequential assignment
• the technique of making the spin-system
assignments, followed by sequence-specific
assignment using unique fragments of sequence, is
known as sequential assignment (Wuthrich)
• there are alternatives to this protocol: one is known
as main-chain directed assignment (Englander).
This technique does not focus on assigning all the
spin systems first. Rather, it focuses on the backbone
and links sizable stretches of backbone residues via
sequential (i,i+1) nOe’s and other nOe’s that are
characteristic of secondary structures. This
technique is particularly useful when there is some
knowledge of secondary structure beforehand.
Close interatomic distances in
secondary structures
parallel beta-sheet
antiparallel
beta-sheet
alpha-helix
type I turn
type II turn
Close interatomic distances in 2ndary
structures
you’ll often see nOe’s associated with secondary structure charted in
this way:
residue #
•a thick bar means a strong nOe (short distance), a thin bar means
a weak nOe (long but still visible distance)
• these sorts of charts allow one to make secondary structure
assignments more or less concurrently with sequential
assignments. As we will see, coupling constants and chemical
shifts also aid in secondary structure assignment
...you can see that it would be easiest to link adjacent residues in
helices with sequential amide-amide nOe’s, whereas in beta sheets
(strand) sequential alpha-amide nOe’s are stronger
d~2.8 Å
d~2.2 Å
Summary of main-chain directed approach
1. assign a few unique
spin systems and use
as entries onto the backbone
Arg Tyr Ser Ala Ala Asn Trp
3. fill in missing spin system
assignments
2. walk down the backbone using
sequential and other backbone nOe’s
“backbone” refers to alpha and amide protons
Summary of sequential approach
1. assign most or all spin systems
Arg Tyr Ser Ala Ala Asn Trp
3. assemble larger sections
of sequence-specific assignments
from dipeptide fragments, until the
whole protein has been assigned
2. connect adjacent spin systems
using backbone nOe’s to identify
unique dipeptides
“backbone” refers to alpha and amide protons
Assignment methods that use
heteronuclear shift correlation
•
•
•
•
for larger proteins (>10-15 kD), assignment methods based on 2D
homonuclear 1H-1H correlation methods (COSY/TOCSY/NOESY) don’t
work very well because of overlapping resonances and broad
linewidths.
an alternative (which is now used even for small proteins in most
cases) is to use heteronuclear shift correlation experiments on 13C, 15N
labelled samples.
in these experiments, magnetization is transferred from 1H to 13C
and/or 15N through large one-bond scalar couplings.
Some relevant scalar coupling constants:
15N-1H
HSQC based techniques
•as we have seen, one of the
simplest types of heteronuclear
shift correlation is the HSQC
experiment, which correlates 1H
chemical shift to the chemical
shift of a 15N or 13C connected by
a single bond
•heteronuclear shift correlation
can be combined with
homonuclear experiments such
as 1H-1H NOESY or TOCSY to
yield 3-dimensional spectra
3D HSQC-NOESY and HSQC-TOCSY
these planes can be thought of as a 15N-1H HSQC
these planes can be thought of
as a 1H-1H NOESY
the 15N shift dimension
can resolve peaks that would
overlap in a 2D NOESY
view of a 3D NOESY experiment
Triple-resonance experiments
•
•
•
•
there is a whole raft of experiments that
use both 13C and 15N correlations to 1H
nuclei
the beauty of these experiments is that
they can connect adjacent residues
without requiring any nOe information-it’s all through-bond scalar coupling
interactions. Makes sequence-specific
assignment more reliable.
they also use mostly one-bond
couplings, which aren’t very sensitive to
the protein conformation (unlike, say,
three-bond couplings, which vary
significantly with conformation, as we
will see)
limiting factors: 13C is expensive and
these exp’ts can be tricky