Resonance assignments Part II: Approaches to sequencespecific assignments

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Resonance assignmentsPart IIApproaches to
sequence-specific assignments
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Sequence-specific assignments

• suppose we have the sequence of our protein from
  some independent measurement
• suppose weve assigned an isoleucine spin system,
  and theres only one isoleucine in the sequence
  (unique), at position 48. Then we know our
  isoleucine is Ile48.
• there wont 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
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Linking spin systems using nOes

• 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,i1) nOe or
  dbN (i,i1) correlation
• daN(i,i1), dNN(i,i1), and sometimes
  dbN(i,i1) are used to connect adjacent residues

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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 weve seen) --gt result is crosspeaks
due to nOe
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2D NOESY linking spin systems
diagonal no magnetization transferred
5.HN/6.HN
4.HN/5.HN
crosspeaks intersection of chemical shifts of
atoms which are close in space, i.e. lt 5 Å
1H
amide-amide region of 2D NOESY of P22 Cro
protein, showing dNN(i,i1) correlations-- can
walk along the chain from one residue to the
next. Residues 3-7 shown.
6.HN/7.HN
3.HN/4.HN
1H
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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,i1) nOes and other nOes 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.

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Summary of sequential approach
1. assign most or all spin systems
Arg Tyr Ser Ala Ala Asn Trp
2. connect adjacent spin systems using backbone
nOes to identify unique dipeptides
3. assemble larger sections of sequence-specific
assignments from dipeptide fragments, until the
whole protein has been assigned
backbone refers to alpha and amide protons
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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
2. walk down the backbone using sequential and
other backbone nOes
3. fill in missing spin system assignments
backbone refers to alpha and amide protons
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Close interatomic distances in secondary
structures
parallel beta-sheet
antiparallel beta-sheet
alpha-helix
type I turn
type II turn
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Close interatomic distances in 2ndary structures
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nOes and secondary structures
residue

• In NMR papers youll 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 nOes 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

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...you can see that it would be easiest to link
adjacent residues in helices with sequential
amide-amide nOes, whereas in beta sheets
(strand) sequential alpha-amide nOes are stronger
d2.8 Å
d2.2 Å
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Modern assignment methods that use heteronuclear
shift correlation

• for larger proteins (gt10-15 kD), assignment
  methods based on the 2D homonuclear 1H-1H
  correlation methods (COSY/TOCSY/NOESY) that weve
  been discussing dont 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.

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Scalar couplings commonly used inheteronuclear
shift correlation
all couplings are in units of Hz
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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

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3D HSQC-TOCSY
CO2-
CH2
H
H
CH2
CH3
C
N
C
N
C
C
O
H
O
H
2 of the dimensions are HN correlation (HSQC) 3rd
dimension is 1H-1H TOCSY correlations from the HN
proton
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3D HSQC-NOESY
CO2-
CH2
H
H
CH2
CH3
C
N
C
N
C
C
O
H
O
H
Like 3D TOCSY but includes interresidue and
interspin system correlations (dashed lines).
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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
NOESY (1H) dimension
the 15N shift dimension can resolve peaks that
would overlap in a 2D NOESY
15N dimension
HN 1H dimension
view of a 3D NOESY experiment
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Analyzing 3D spectra
rather than try to look at this whole thing
at once
NOESY or TOCSY (1H) dimension
look at vectors or strips corresponding
to peaks on an HSQC (particular 15N and HN shift
combinations)--gt NOESY/TOCSY correlations will
be along the length of the strip
15N dimension
HN 1H dimension
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Extracting strips in a 3D
Strip of 3D corresponding to peak in HSQC
F2 120 ppm (plane of paper)
Use 2D HSQC as reference spectrum
0 ppm
F2120 ppm, F3 8.0 ppm
crosspeaks to side chain 1H
15N (F2 in 3D)
F1 NOESY or TOCSY dimension
8.1-7.9
crosspeak to alpha
diagonal peak (amide region)
HN (F3 in 3D)
10 ppm
want to look at TOCSY or NOESY correlations from
the amide proton corresponding to this HSQC peak
8.1-7.9 ppm
HN dimension (F3)
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Classifying side chains in 3D TOCSY
0 ppm
0 ppm
0 ppm
b
b
g
b
a
a
a
5 ppm
5 ppm
5 ppm
pair of betas around 3 ppm aromatic (YHWF) or
Asp/Asn (DN)
single pk in alpha region plus single peak 1-2
ppm probable Ala (A)
set of 4 peaks in 1.9-2.6 region Gln, Glu, Met
(QEM)
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Using 3D TOCSY/NOESY dual strip analysis
3D TOCSY
3D NOESY
b
daN(i,i1)
b
dbN(i,i1)
g
same residue
b
a
different residue
a
(YHWHDN)
(EQM)
A
(YHWHDN)
(EQM)
A
TOCSY --gt intraresidue xpks 1. spin system
classifications
NOESY --gt interresidue xpks--gt 2. connect strips
into sequence fragments
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3. take fragment from strip analysis...match
(EQM)A(YHWFDN) pattern to your protein sequence...

• MQTLSERLKKRRIALMTQTELAVKQQ
• SIQLIEAYVTKRPRFLFEIAMALNCDPV
• WLQYGTKRGKAA
• only the E32-Y34 fragment matches...
• sequence specifically assign
• strips in the fragment to
• E32, A33 and Y34.

(EQM)
A
(YHWFDN)
E32
A33
Y34
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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...
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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--its all through-bond scalar
  coupling interactions. Makes sequence-specific
  assignment more reliable.
• they also use mostly one-bond couplings, which
  arent 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
  expts can be tricky

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Beyond spin systems connecting residues using
heteronuclear J couplings
-7 Hz
11 Hz
H
H
H
H
H
H
C
C
C
N
C
C
N
C
N
O
R
R
O
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