Download Sequence-specific assignments

Resonance assignments
Part II:
Approaches to sequence-specific
assignments
Sequence-specific assignments
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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 (or tripeptide etc...)
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
The 2D NOESY pulse sequence
from
Glasel &
Deutscher
p. 354
mixing period tm between t1 and t2 allows for
cross-relaxation between nuclei
(mostly zero quantum as we’ve seen)
--> result is crosspeaks due to nOe
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
amide-amide region of
2D NOESY of
P22 Cro protein,
showing dNN(i,i+1)
correlations-can “walk” along the
chain from one
residue to the next.
Residues 3-7 shown.
Classic 1H resonance assignment
protocols
• Sequential assignment method (Wuthrich)
A method in which one first makes the spin-system
assignments, followed by sequence-specific assignment using
unique fragments of sequence.
• Main-chain directed assignment method (Englander).
This alternative 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 (more on this in a second). This technique is
particularly useful when there is some knowledge of secondary
structure beforehand.
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
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
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
nOes and secondary structures
residue #
• In NMR papers you’ll sometimes see charts like the one shown above. A
thick bar means a strong nOe (short distance), a thin bar means a weak
nOe (long but still visible distance)
• The fact that certain nOe’s are characteristic of secondary structures
allows one to make secondary structure assignments more or less
concurrently with sequential assignments. As we will soon 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
Modern assignment methods that
use heteronuclear shift correlation
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for larger proteins (>10-15 kD), assignment methods based on the 2D
homonuclear 1H-1H correlation methods (COSY/TOCSY/NOESY) that
we’ve been discussing 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 between 1H, 13C and
/or 15N through large one-bond or in some cases two-bond scalar
couplings.
Scalar couplings commonly used in
heteronuclear shift correlation
all couplings are in units of Hz
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
•2D heteronuclear shift
correlation can be combined with
homonuclear experiments such
as 1H-1H 2D NOESY or 2D
TOCSY to yield 3-dimensional
spectra
3D HSQC-TOCSY
CO2CH2
H
CH2
N
C
C
H
O
H
CH3
N
C
C
H
O
2 of the dimensions are HN correlation (HSQC)
3rd dimension is 1H-1H TOCSY correlations from the HN proton
3D HSQC-NOESY
CO2CH2
H
CH2
N
C
C
H
O
H
CH3
N
C
C
H
O
Like 3D TOCSY but includes interresidue and interspin system
correlations (dashed lines).
3D HSQC-NOESY and HSQC-TOCSY
these planes can be thought of as a 15N-1H HSQC
the planes (parallel to the slide)
can be thought of as a 1H-1H
NOESY
(1H)
NOESY
dimension
the 15N shift dimension can resolve
peaks that would overlap in a 2D NOESY
15N
dimension
HN 1H
view of a 3D NOESY experiment
dimension
Analyzing 3D spectra
rather than try to look
at this whole thing at
once
NOESY or
TOCSY
(1H)
dimension
15N
dimension
HN 1H
dimension
look at
vectors or “strips”
corresponding to
peaks on an HSQC
(particular 15N and HN
shift combinations)-->
NOESY/TOCSY
correlations will be along
the length of the strip
Extracting strips in a 3D
Use 2D HSQC as reference spectrum
Strip of 3D corresponding
to peak in HSQC
0 ppm
F2:120 ppm, F3: 8.0 ppm
15N
(F2
in
3D)
F2 = 120 ppm
(plane of paper)
crosspeaks to
side chain 1H
F1:
8.1-7.9
crosspeak to
alpha
NOESY or
TOCSY
dimension
diagonal peak
(amide region)
HN (F3 in 3D)
want to look at TOCSY or NOESY
correlations from the amide proton
corresponding to this HSQC peak
10 ppm
8.1-7.9 ppm
HN dimension (F3)
Classifying side chains in 3D TOCSY
0 ppm
0 ppm
0 ppm
b
b
g
b
a
a
a
5 ppm
set of 4 peaks in
1.9-2.6 region:
Gln, Glu, Met
(QEM)
5 ppm
5 ppm
single pk in alpha
pair of betas
region plus single
around 3 ppm:
peak 1-2 ppm:
aromatic (YHWF)
probable Ala (A)
or Asp/Asn (DN)
Using 3D TOCSY/NOESY dual strip analysis
3D TOCSY
3D NOESY
b
daN(i,i+1)
b
dbN(i,i+1)
g
same
residue
b
a
different
residue
a
(EQM)
A
(YHWHDN)
TOCSY --> intraresidue xpks
1. spin system classifications
(EQM)
A
(YHWHDN)
NOESY --> interresidue xpks-->
2. connect strips into sequence fragments
3. take fragment from strip
analysis...match (EQM)A(YHWFDN)
pattern to your protein sequence...
MQTLSERLKKRRIALMTQTELAVKQQ
SIQLIEAYVTKRPRFLFEIAMALNCDPV
WLQYGTKRGKAA
only the E32-Y34 fragment matches...
4. sequence specifically assign
strips in the fragment to
E32, A33 and Y34.
(EQM)
A
(YHWFDN)
E32
A33
Y34
5. annotate the corresponding 2D HSQC peaks
with the new assignments
E32
A33
15N
(F2
in
3D)
Y34
HN (F3 in 3D)
6. proceed until entire HSQC is assigned...
Triple-resonance experiments
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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
Beyond “spin systems”: connecting
residues using heteronuclear J couplings
-7 Hz
H
H
N
C
C
R
O
11 Hz
H
H
N
C
C
R
O
H
H
N
C
C
R
O
the HNCA experiment above connects the HN group to the alpha
carbon of both the same residue and the previous one. The
two-bond N-C coupling traverses the carbonyl group, which is
a barrier to using 1H-1H scalar couplings to connect residues