What is the NMR Assignment Issue

1
NMR Assignments

• What is the NMR Assignment Issue?
• Each observable NMR resonance needs to be
  assigned or associated with the atom in the
  protein structure.
• NMR spectra of proteins are complex, where the
  complexity increases with the size or number of
  residues of the protein
• Use 13C 15N isotope enrichment to simplify the
  NMR spectra need to assign these NMR resonances
• a typical protein will have hundreds of 1H, 13C
  and 15N NMR resonance to assign

Protein PDB File
1H NMR Spectra
2
NMR Assignments
Again, as illustrated here, the goal is to
explicitly assign each H, C, N in the proteins
primary sequence with its corresponding NMR
resonance
15N 114.8 ppm HN 7.08 ppm
15N 125.6 ppm HN 8.20 ppm
13Ca 58.6 ppm Ha 4.09 ppm
15N 119.3 ppm HN 7.76 ppm
13Ca 55.5 ppm Ha 3.76 ppm
13CO 171.9 ppm
13CO 178.1 ppm
13CO 170.9 ppm
13Cb 17.5 ppm Hb 1.45 ppm
13Cb 42.9 ppm Hb 1.52 ppm
13Cb 64.8 ppm Hb 3.73 ppm
13Cg 27.9 ppm Hg 1.65 ppm
13Ca 59.9 ppm Ha 4.35 ppm
13Cd 25.4 ppm 25.7 ppm Hd 0.82 ppm 0.98 ppm
3
NMR Assignments

• How Are NMR Assignments Made For a Protein?
• Requires the collection and analysis of
  multidimensional NMR data
• 2D, 3D, 4D NMR spectra
• This in turns requires software to assist in the
  processing and analysis of the data
• ongoing effort to develop software to automate
  NMR assignments
• not 100 efficient but significantly aids in
  the manual assignment

Assignment Table
. . .
4
NMR Assignments

• NMR Data Processing Software
• Needs to specifically handle format of
  multidimensional NMR data
• 2D, 3D, 4D NMR spectra
• NMRPipe, Felix, ACD and others
• all have similar functions and capability
• all handle common instrument data formats
  (Bruker, Varian)
• choice is primarily based on personal preference

• NMRpipe
• UNIX/LINUX
• simple script to process NMR data
• mimics flow of processing steps
• uses UNIX pipe functionality to pass data
  between one function to the next

5
NMR Assignments

• NMR Data Processing Software
• Main steps in the processing process include
• window function (SP), zero fill (ZF), Fourier
  transform
• (FT), phase (PS), transpose (TP)
• Other steps include
• removing solvent (SOL), linear prediction (LP)
  and data
• extraction (EXT)
• These steps are simply repeated for each
  dimension of the
• NMR data

Standard Processing Script for 3D NMR Data
X
Processing steps for X,Y,Z dimensions of 3D
spectra
Y
Z
6
NMR Assignments

• NMR Data Processing Software
• Because of the exponential increase in time to
  collect nD NMR spectra, the number of data points
  collected for the indirect FIDs are kept to a
  minimum
• 1D NMR few mins. ? 2D few hours ? 3D few
  days
• 1D NMR 8-32K pts ? 2D 2K x 512 pts ? 3D 2K x 128
  x 80 pts
• Two major impacts
• FIDs in indirect dimension are typically
  truncated ? artifacts in the spectra
• FIDs in indirect dimension have very low
  resolution
• These issues are addressed in processing the
  data
• ZF, SP, LP

FT
7
NMR Assignments

• NMR Data Processing Software
• A main goal in applying a window function for a
  nD NMR spectra is to remove the truncation by
  forcing the FID to zero.

Truncated FID with spectra wiggles
Apodized FID removes truncation and wiggles
8
NMR Assignments

• NMR Data Processing Software
• Some common window functions with the
  corresponding NMRPipe command

9
NMR Assignments

• NMR Data Processing Software
• Improve digital resolution by adding zero data
  points at end of FID
• essential for nD NMR data
• no significant gain after one ZF, just
  interpolation between points

8K data
8K zero-fill
8K FID
16K FID
No zero-filling
8K zero-filling
10
NMR Assignments

• NMR Data Processing Software
• Linear Prediction
• extrapolate FID data in time domain
• enhances resolution
• works best for data without significant
  relaxation
• assumes sinusoid shape
• a set of coefficients is found such that linear
  combination of a group of points predicts the
• next point in the series.
• number of coefficients determine the number of
  NMR signals (damped sinusoids) that
• can be predicted
• LP is usually limited to extending data to about
  twice its original size
• forward linear prediction - points immediately
  after each group are predicted
• backward linear prediction - points immediately
  before each group are predicted
• forward-backward linear prediction - combines
  results from separate forward- and
• backward-linear prediction calculations.

LP
11
NMR Assignments

• NMR Data Processing Software
• Linear Prediction
• model (set of coefficient) can be applied to
  predict a new synthetic point
• uses a group of existing points from the
  original data
• new point along with group from the original
  data is used to predict yet another point
• process can be continued indefinitely
• becomes unstable when group contains all
  synthetic points
• Mirror Image LP
• LP order (number of coefficients) must be as
  large as the number of signals to extract,
• but smaller than half the original data size.
• For constant time data, (no decay) can
  temporarily add the data's mirror image
• complex conjugate for the LP calculation and
  then discard it.
• time increment must be the same between each
  point
• either 0,0 or 90,-180 phase correction

LP
Progress in Nuclear Magnetic Resonance
Spectroscopy (1988), 20(6),515-626
12
NMR Assignments

• NMR Data Processing Software
• Effects of Combining Linear Prediction with Zero
  Filling
• significant improvement in resolution for nD NMR
  data collected with minimal data points

13
NMR Assignments

• NMR Data Processing Software
• Solvent Removal (SOL)
• protein NMR spectra are typical collected in
  water
• the large solvent signal can interfere with the
  interpretation of the NMR data
• Carrier frequency is usually centered on the
  water signal
• the siganl associated with the water resonacne
  can be filtered or subtracted from the time
  domain of the FID

SOL
14
NMR Assignments

• NMR Data Processing Software
• Solvent Removal (SOL)

without Solvent Subtraction
with Solvent Subtraction
15
NMR Assignments

• NMR Data Processing Software
• Phase Correction (PS)
• Because of the challenges of phasing nD NMR data
  and the baseline artifacts that first-order phase
  corrections are known to cause, typically phase
  corrections are set to 0,0 or 90-180 by proper
  delays in the pulse sequence
• A number of methods of data collection are used
  to obtain phase correction in the indirect
  dimensions
• Fourier transformed data contains a real part
  that is an absorption lorentzian and an
• imaginary part which is a dispersion
  lorentzian
• we want to maintain the real absorption mode
  line-shape
• done by applying a phase factor (exp(iQ)) to set
  F to zero
• this is what we are doing when we phase the
  spectra

16
NMR Assignments

• NMR Data Processing Software
• Phase Correction (PS)
• Phase of the peak is determined by the relative
  phase of the pulse and the receiver
• to obtain correct phasing in the indirect
  dimension, we need to collect both sine and
  cosine modulated data
• alternate both the phase of the pulse relative
  to the receiver and the storage of this data
  between real (sine) and imaginary (cosine)

17
NMR Assignments

• NMR Data Processing Software
• Phase Correction (PS)
• Phase of the peak is determined by the relative
  phase of the pulse and the receiver
• Also determines the order in which the data is
  stored.
• Some Common Phase Cycle Schemes
• STATES phase cycles the 90o-pulses prior to t1
  incrimination by 900
• TPPI phase cycles both the receiver and the
  90o-pulses prior to t1 by 90o for each t1
• increment
• States-TPPI phase cycles both the receiver and
  the 90o-pulses prior to t1 by 180o for
• each t1 increment
• Echo-antiecho uses gradients to reduce the
  number of phase cycling steps and
• combines N (echo) and P(antiecho) coherence
  selection

18
NMR Assignments

• NMR Data Processing Software
• Phase Correction (PS)

19
NMR Assignments

• NMR Data Processing Software
• Phase Correction (PS)

The phase introduced by a gradient of duration tG
to coherence of order p which involves k spins
with gyromagnetic ratios gk is given by f(r)
r Gz tG Sk( pk?k)
Complex Fourier transformation and combination of
the two signals yields a purely absorptive
spectrum with frequency sign discrimination.
20
NMR Assignments

• NMR Data Processing Software
• Data Conversion (bruk2pipe)
• Prior to processing the NMR data by NMRPipe is a
  requirement to convert the file format
• This process requires defining some important
  experimental parameters
• number of points, sweep width, phase cycling,
  etc

Phase cycling determines how the data is stored
and retrieved
States - odd data points are written to the real
data array, even data points to the imaginary
data array. source 1 2 3 4 real 1 3
imaginary 2 4 TPPI - data are copied to the real
data array. source 1 2 3 4 real 1 2 3
4 Echo-antiecho - 4 data points are mixed and
written to the real and imaginary data arrays.
source 1 2 3 4 real 13 4-2 imaginary 24
1-3 States-TPPI - Same as States, but every
second real and imaginary data point is
multiplied by -1. source 1 2 3 4 real 1 -3
imaginary 2 -4
21
NMR Assignments

• NMR Data Processing Software
• NMR data analysis/visualization
• NMRDraw, NMRView, PIPP, etc
• Again, most programs have similar functionality,
  choice is based on personal preference
• display the data (zoom, traces, step through
  multiple spectra, etc)
• Peak-picking identify the X,Y or X,Y,Z or
  X,Y,Z,A chemical shift coordinate positions for
  each peak in the nD NMR spectra

Peak Picking List
22
NMR Assignments

• NMR Data Processing Software
• NMR data analysis/visualization
• Peak Picking
• Critical for obtaining accurate NMR assignments
• Especially for software for automated
  assignments
• Only provide primary sequence and peak-pick
  tables
• Two General Approaches to Peak Picking
• Manual
• time consuming
• can evaluate crowded regions more
• effectively
• Automated
• pick peaks above noise threshold
• OR
• pick peaks above threshold with
• characteristic peak shape
• only about 70-80 efficient
• crowded overlap regions and noise
• regions (solvent, T2 ridges) cause problems

J. OF MAG. RES. 135, 288297 (1998)
23
NMR Assignments

• NMR Data Processing Software
• Automated NMR assignments
• AutoAssign, CONTRAST, GARANT, PASTA, etc
• uses peak lists, primary protein sequence,
  details of NMR experiments
• tries to mimic skilled user, uses databases of
  previous assignments, etc
• Automated analysis of NOESY data is a sub-set of
  the NMR assignment issue with programs designed
  to specifically address this need
• AutoStructure, CANDID, ARIA, ROSSETTA, etc

From, peak-lists and protein sequence, software
attempts to make the assignment. Not 100
success rate, still need user intervention to
complete/correct assignments. Most problems
arise from quality of peak-list noise, missing
peaks, etc. Need to Know How Assignments are
Made!
24
NMR Assignments

• NMR Assignment Protocol
• 2D NMR Experiments
• Kurt Wüthrich Nobel prize in 2002 for developing
  NMR to determine 3D structures of proteins.
• Wüthrich NMR of Proteins and Nucleic Acids
  1986, John Wiley Sons
• Applicable for proteins of lt100 amino acids
• Primarily dependent on three 2D experiments
  NOESY, COSY, TOCSY
• Sequence-Specific Resonance Assignments in
  Proteins (Backbone Assignemnts)

Takes advantage of short sequential distances
between CaiH, CbiH and NHi1
25
NMR Assignments

• 2D NMR Experiments
• 2D COSY
• Correlation Spectroscopy
• Correlates 1H resonances that are scalar coupled
  (3J)
• Identifies which NHi resonances are bonded to
  CaHi resonances
• separated by three-bonds
• chemical shift evolution based on J occurs
  during t1
• requires the sample be in H2O (90/10 H2O/D2O) to
  observe NH
• all three-bond couplings observed, not just
  NH-Ca
• spectra is symmetric
• strength of cross peak depends on strength of
  coupling constants
• all predicted peaks are not necessarily observed
• weak couplings
• obscured by solvent, noise
• overlap or degenerate peaks

26
NMR Assignments

• 2D NMR Experiments
• 2D COSY
• Typical Small Protein COSY

27
NMR Assignments

• 2D NMR Experiments
• 2D NOESY
• Nuclear Overhauser Spectroscopy
• Correlates 1H resonances that close in space
  (5Å)
• also contains COSY peaks
• NOE intensity builds up during mixing time (tm),
  ususally 100-150 ms
• Correlates NHi1 resonances with CaHi resonances

28
NMR Assignments

• 2D NMR Experiments
• 2D NOESY
• Typical Protein NOESY (Lysozyme)

Both NHi-Cai and NHi1-Cai are present
29
NMR Assignments

• 2D NMR Experiments
• Making the Sequential Assignments
• Connecting COSY (NHi-Cai) peaks with NOESY
  (NHi1-Cai)
• COSY experiment allows you to identify the
  NHi-Cai cross peaks in the NOESY
• experiment
• N-terminal amino acid only has one cross peak
  associated with its NH chemical shift

The Backbone Walk
NOESY cross peak
COSY cross peak
NHi1-Cai
A24
NHi-Cai
NHi-Cai
NHi1-Cai
T27
Y28
NHi-Cai
NHi1-Cai
F25
NHi-Cai
D26
NHi1-Cai
NHi-Cai
T27
D26
F25
A24
Y28
Biochemistry 1989, 28, 1048-1054
NH Chemical Shifts (ppm)
30
NMR Assignments

• 2D NMR Experiments
• Verifying the Sequential Assignments and
  Side-Chain Assignments
• The accuracy of the backbone assignments from
  connecting COSY (NHi-Cai) peaks with
• NOESY (NHi1-Cai) can be verified by proper
  assignment of the side-chain with the
• backbone assignments.
• know the primary sequence of the protein
• therefore, know what amino acid is residue (i)
  and what amino-acid should be (i1)
• amino acid type indicates the number and type
  or chemical shifts that should be observed for
  the residue

As example Gly no side chain Ala single
methyl (1.39 ppm) Val two g methlys (0.97
0.94 ppm) one Hb (2.13 ppm)
31
NMR Assignments

• 2D NMR Experiments
• Connectivity Patterns
• COSY TOCSY patterns
• for the 20 amino acids
• Side-chain assignments
• involves matching
• the expected patterns
• and typical chemical
• shift ranges
• Some connectivity
• patterns are not unique
• and can only eliminate
• some possible
• assignments

In real data, overlapping or missing cross-peaks
are common. Connectivity pattern may not exactly
match predicted.
32
NMR Assignments

• 2D NMR Experiments
• Connectivity Patterns

Leu - expected
Cd
Cb
Cg
Ca
Leu - actual
Cb/Cg
Cb
Cd
Ca
Structure induces chemical shift changes which
perturbs the pattern and induces overlap. But,
the data has to be consistent with the amino-acid
spin system or the assignment is probably
incorrect
33
NMR Assignments

• 2D NMR Experiments
• Connectivity Patterns
• NMR assignments should be consistent with
  expected trends
• significant differences should be explained by
  the structure
• (ring current, h-bonds, etc)

34
NMR Assignments

• 2D NMR Experiments
• 2D TOCSY
• TOtal Correlation SpectroscopY
• cross peaks are generated between all members of
  a coupled spin network
• NMR resonances for the complete side-chain spin
  systems is obtained
• coherence transfer period occurs during a
  multi-pulse spin-lock period
• length of spin-lock determines how far the
  spin coupling network will be probed
• 1/(10 JHH) should be used for each transfer step
• not all correlations are observed

COSY
TOCSY
Spin-Lock Pulse (14ms)
35
NMR Assignments

• 2D NMR Experiments
• 2D TOCSY

• What happens during the spin-lock time cannot be
  described in terms of vector models or product
• operators, because it relies on strong
  coupling
• Under strong coupling, chemical shift
  differences between different spins become
  negligible
• Two states ab and ba become identical in energy
• Instead of transition of single spins, the
  coherences now involves transitions of
  combinations
• of spins
• Under this condition, a coherence of one spin is
  actually in resonance with a coherence of its
• coupling partner(s) (all with the same
  frequency), and will oscillate back and forth
  between
• all coupled spins

36
NMR Assignments

• 2D NMR Experiments
• 2D TOCSY
• Typical Small Protein TOCSY
• Side-chain spin systems are
• correlated with NH resonance

Boxed regions indicate side-chain spin systems
for His and Ile, respectively
Bull. Korean Chem. Soc. 2001, Vol. 22, No. 5 507
37
NMR Assignments

• 3D NMR Experiments
• Takes advantage of 13C and 15N labeling
• Extends assignments to proteins in the 20-25
  kDa range
• Extends Connectivity by Scalar Coupling (J)
  into 3D dimensions
• Primarily uses one-bond heteronuclear coupling
  (1H-13C, 1H-15N)
• 1J generally stronger than 3J
• 2D 1H-15N HSQC is the root experiment of most of
  the standard triple-resonance (1H, 13C, 15N) NMR
  experiments
• 3D NMR simplifies data and removes overlap by
  spreading information into third dimension
• Requires multiple experiments ( 6) to walk
  through the backbone assignments similar to the

38
NMR Assignments

• 3D NMR Experiments
• 2D 1H-15N HSQC experiment
• correlates backbone amide 15N through one-bond
  coupling to amide 1H
• in principal, each amino acid in the protein
  sequence will exhibit one peak in the 1H-15N
• HSQC spectra
• also contains side-chain NH2s (ASN,GLN) and NeH
  (Trp)
• position in HSQC depends on local structure and
  sequence
• no peaks for proline (no NH)

Side-chain NH2
39
NMR Assignments

• 3D NMR Experiments
• Consider a 3D experiment as a collection of 2D
  experiments
• z-dimension is the 15N chemical shift
• 1H-15N HSQC spectra is modulated to include
  correlation through coupling to a another
  backbone atom
• All the 3D triple resonance experiments are then
  related by the common 1H,15N chemical shifts of
  the HSQC spectra
• The backbone assignments are then obtained by
  piecing together all the jigsaw puzzles pieces
  from the various NMR experiments to reassemble
  the backbone

40
NMR Assignments

• 3D NMR Experiments
• Amide Strip

3D cube
amide strip
2D plane
Strips can then be arranged in backbone
sequential order to visual confirm assignments
41
NMR Assignments

• 3D NMR Experiments
• 3D HNCO Experiment
• common nomenclature ? letters indicate the
  coupled backbone atoms
• correlates NHi to Ci-1 (carbonyl carbon, CO or
  C)
• no peaks for proline (no NH)
• Like the 2D 1H-15N HSQC spectra, each amino
  acid should display a single peak in the 3D HNCO
  experiment
• identifies potential overlap in 2D 1H-15N HSQC
  spectra, especially for larger MW proteins
• most sensitive 3D triple resonsnce experiment
• may observe side-chain correlations

1JNC
1JNH
42
NMR Assignments

• 3D NMR Experiments
• 3D HNCO Experiment

43
NMR Assignments
slice through 3D cube

• 3D NMR Experiments
• 3D HNCO Experiment

One expanded plane or slice from a 3D HNCO
experiment, where the 15N chemical shift is
118.21 ppm A total of 128 planes, with a digital
resolution of 0.28 ppm per plane for the entire
experiment.
44
NMR Assignments

• 3D NMR Experiments
• 3D HN(CA)CO Experiment
• correlates NHi to COi
• relays the transfer through Cai without chemical
  shift evolution
• uses stronger one-bond coupling
• contains only intra correlation
• provides a means to sequential connect NH and CO
  chemical shifts
• match NHi-COi (HN(CA)CO with NHi-COi-1 (HNCO)
• not sufficient to complete backbone assignments
  because of overlap and
• missing information
• every possible correlation is not observed
• need 2-3 connecting inter and intra correlations
  for unambiguous
• assignments
• no peaks for proline (no NH) breaks assignment
  chain
• but can identify residues i-1to prolines

1JNCa
1JCaC
1JNH
45
NMR Assignments

• 3D NMR Experiments
• 3D HN(CA)CO Experiment

46
NMR Assignments

• 3D NMR Experiments
• 3D HN(CA)CO Experiment

HNCO and HN(CA)CO pair for one residues NH
Connects HNi-COi with HNi-COi-1
Amide Strips from the 3D HNCO and HN(CA)CO
experiments arranged in sequential order
Journal of Biomolecular NMR, 9 (1997) 1124
47
NMR Assignments

• 3D NMR Experiments
• 3D HNCA Experiment
• correlates NHi to Cai-1 and Cai
• typically the intensity of NHi-Cai gt NHi-Cai-1,
  1JNCa gt 2JNCa
• NHi-Cai-1 correlation not always seen
• could be weak or degenerate with NHi-Cai
• contains both inter and intra correlations
• provides a means to sequential connect NH and Ca
  chemical shifts
• not sufficient to complete backbone assignments
  because of overlap
• need 2-3 connecting inter and intra correlations
  
• no peaks for proline (no NH) breaks assignment
  chain
• but can identify residues i-1to prolines

2JNCa
1JNCa
1JNH
48
NMR Assignments

• 3D NMR Experiments
• 3D HNCA Experiment

49
NMR Assignments
Amide Strips from the 3D HNCA experiment
arranged in sequential order

• 3D NMR Experiments
• 3D HNCA Experiment

Cai-1
Correlation of the Cai and Cai-1 sequentially
aligns the two NHs in the proteins sequence.
Cai
Each strip corresponds to one NH resonance in a
given 15N plane
J. of Biomol. NMR, 14 8588, 1999.
50
NMR Assignments

• 3D NMR Experiments
• 3D HN(CO)CA Experiment
• correlates NHi to Cai-1
• relays through 1JNC without chemical shift
  evolution
• NHi-Cai-1 correlation is more sensitive than
  HNCA experiment
• unambiguous NHi-Cai-1 assignments
• avoids possible overlap in HNCA experiment
• companion experiment to HNCA
• provides a means to sequential connect NH and Ca
  chemical shifts
• NHi-Cai (HNCA) matches with NHi-Cai-1 (HN(CO)CA)
  
• not sufficient to complete backbone assignments
  because of overlap
• need 2-3 connecting inter and intra correlations
  
• no peaks for proline (no NH) breaks assignment
  chain
• but can identify residues i-1to prolines

1JCCa
1JNC
1JNH
51
NMR Assignments

• 3D NMR Experiments
• 3D HN(CO)CA Experiment

52
NMR Assignments

• 3D NMR Experiments
• 3D HN(CO)CA Experiment

one residues NH
HNCA
HN(CO)CA
NHi-Cai
NHi-Cai-1
Journal of Biomolecular NMR, 9 (1997) 167180
53
NMR Assignments

• 3D NMR Experiments
• 3D CBCANH Experiment
• correlates NHi to Cai, Cai-1 and Cbi, Cbi-1
• transfer is simultaneously started on Ha Hb
  (both i and i-1)
• typically the intensity of NHi-Cai NHi-Cbi gt
  NHi-Cai-1 NHi-Cbi-1
• 1JNCa gt 2JNCa
• can usually distinguish Ca from Cb from chemical
  shift difference
• NHi-Cai NHi-Cai-1 are opposite sign of NH-Cbi
  NH-Cai-1
• one set of peaks are positive intensity and the
  other set is negative
• only Gly NHi-Cai-1 NHi-Cai correlations are
  seen
• contains both intra and inter correlations
• provides a means to sequential connect NH, Ca
  and Cb chemical shifts
• the 2 connections of inter and intra
  correlations may be sufficient to unambiguously
• assign the backbone
• weakest experiment, so all the necessary data is
  usually not present and the single
• experiment is typically inadequate to assign
  the complete backbone

Match-up the intra and inter correlations
1JNCa
1JNCb
2JNCa
1JNH
54
NMR Assignments

• 3D NMR Experiments
• 3D CBCANH Experiment

55
NMR Assignments
Amide Strips from the 3D CBCANH experiment
arranged in sequential order

• 3D NMR Experiments
• 3D CBCANH Experiment

Correlation of the Cbi and Cbi-1 sequentially
aligns the two NHs in the proteins sequence.
Correlation of the Cai and Cai-1 sequentially
aligns the two NHs in the proteins sequence.
Note contours of opposite intensity are shown in
different colors
IUBMB Life, 52 291302, 2001
56
NMR Assignments

• 3D NMR Experiments
• 3D CBCA(CO)NH Experiment
• correlates NHi to Cai-1 and Cbi-1
• can usually distinguish Ca from Cb from chemical
  shift difference
• sometimes NHi-Cai-1 and NHi-Cbi-1 may be
  oppositely phased
• one peak positive intensity the other negative
• only Gly NHi-Cai-1 correlations are seen
• no peaks for proline (no NH) breaks assignment
  chain
• transfer is started on simultaneously on Hai-1,
  Hbi-1, relayed through CO without
• chemical shift evolution (1JCaC, 1JCN)
• contains only inter correlations
• provides a means to sequential connect NH, Ca
  and Cb chemical shifts with a
• companion experiment (s)
• companion experiments would provide NHi-Cai
  (HNCA) and NHi-Cbi
• (CBCANH)
• the 2 connections of inter and intra
  correlations may be sufficient to
• unambiguously assign the backbone

1JCCb
1JCaC
1JNH
57
NMR Assignments

• 3D NMR Experiments
• 3D CBCA(CO)NH Experiment

58
NMR Assignments

• 3D NMR Experiments
• 3D CBCA(CO)NH Experiment

Correlation of the Cai Cai-1 and Cbi Cbi-1
sequentially aligns each pair of NHs in the
proteins sequence.
Amide Strips from the 3D CBCANH (right) and
CBCA(CO)NH (left) experiment arranged in
sequential order
Journal of Biomolecular NMR, 10 (1997) 7788
59
NMR Assignments
60
NMR Assignments

• 3D NMR Experiments
• Typically collect 1024 x 64 x 40 complex points
  in
• each dimension
• Typical digital resolution is 0.02ppm (1H) x
  0.15
• ppm (13C) x 0.28 ppm (15N)
• resolution is better in some experiments that
  require smaller sweep-width.
• need to allow for significant error when
  comparing chemical shift values from different
  NMR experiments
• conservative? use twice digital resolution
• Typical experiment time is 2.5 days

61
NMR Assignments
62
NMR Assignments

• 3D NMR Experiments
• Large Variety of Experiments Based on These 3D
  Triple Resonance Experiments
• Proton Versions of the Experiments
• CBCA(CO)NH ? HBHA(CO)NH
• HNCA ? HNHA
• CBCANH ? HBHANH
• provides even more possible i i-1 types of
  correlations
• more confirmed observed correlations more
  definitive the assignment
• Modifications are constantly being made and new
  versions or variations are
• constantly described in the literature to
  improve sensitivity and eliminate artifacts
• constant time, gradient enhancements,
  out-and-back, cryoprobe versions, etc
• Specific modifications to handle larger
  molecular-weight proteins
• deuterium decoupling ? deuterated proteins
• TROSY versions

63
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• Need to correlate all the information from all
  the available experiments
• Cai ? Cai-1
• Cbi ? Cbi-1
• COi ? COi-1
• Hai ? Hai-1

Journal of Biomolecular NMR, 9 (1997) 167180
64
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• The process is a multi-step approach
• (1) correlate all the experimental data with each
  NH root observed in the 2D 1H-15N HSQC spectra

65
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• The process is a multi-step approach
• (2) Match pairs of NH roots based on i and i-1
  correlations

66
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• The process is a multi-step approach
• (3) Extend pairs of NH roots and match to protein
  primary sequence

Identify overlapping spin-system pairs
connect spin-system pairs
67
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• The process is a multi-step approach
• (3) Extend pairs of NH roots and match to protein
  primary sequence

Identify possible residue types by chemical shift
ranges
68
NMR Assignments

• 3D NMR Experiments
• Backbone Assignments
• The process is a multi-step approach
• (3) Extend pairs of NH roots and match to protein
  primary sequence

Find potential match in sequence
Make assignment
69
NMR Assignments

• 3D NMR Experiments
• Side-chain Assignments
• Help confirm the backbone assignment
• Similar in principal to 2D assignment approach
• Correlate entire spin-system with NH backbone
• Use TOCSY to observe entire spin-system
• CC(CO)NH HCC(CO)NH
• Relay magnetization from NH through side-chain
  carbon or hydrogen chemical
• shifts
• Start simultaneously on all side-chain hydrogens
• Also, overlap with Ca and Cb chemical shifts
  from other triple-resonance
• experiments to confirm side-chain assignments

70
NMR Assignments
CC(CO)NH
Which Hs match the Cs?
HCC(CO)NH

• 3D NMR Experiments
• Side-chain Assignments
• CC(CO)NH HCC(CO)NH
• Can assign residue type by the number of
• observed resonances and the chemical shift
• ranges
• may be able to assign Cg, Cd, Ce from
• chemical shift values and from
• previously assigned Ca and Cb
• less likely to assign Hg, Hd and He,
• unless unique chemical shift
• need companion experiments to connect
• carbon and hydrogen chemical shifts.

d1
g2
g1
b
a
Biochemistry, Vol. 34, No. 42, 1995
71
NMR Assignments

• 3D NMR Experiments
• Side-chain Assignments
• HCCH-TOCSY HCCH-COSY
• relays magnetization from side-chain and
  backbone 1H 13C via coupling
• constants
• Experiments have symmetry
• 1Ha-13Ca diagonal shows cross peak to 1Hb
• AND
• 1Hb-13Cb diagonal shows cross peak to 1Ha
• does not correlate to backbone NH? no direct
  connection with other triple-
• resonance experiments
• sample can be collected in D2O

72
NMR Assignments

• 3D NMR Experiments
• Side-chain Assignments
• HCCH-TOCSY
• HCCH-COSY

Slices taken from different 13C chemical shift
planes at different 1H chemical shifts
illustrates the entire spin system for a single
side-chain
Symmetry each HCd shows a cross peak to Ha and
the HCa shows a crosspeak to both HCd
Note Symmetry peaks may not always be present
(separate pathways, separate relative
sensitivity). Presence of a symmetry peak
increase the likelihood of correct assignment
Journal of Biomolecular NMR, 9 (1997) 445446
73
NMR Assignments

• 4D NMR Experiments
• Consider a 4D NMR experiment as a
• collection of 3D NMR experiments
• still some ambiguities present when correlating
  multiple 3D triple-resonance experiments
• 4D NMR experiments make definitive sequential
  correlations
• increase in spectral resolution
• Overlap is unlikely
• loss of digital resolution
• need to collect less data points for the 3D
  experiment
• If 3D experiment took 2.5 days, then each 4D
  time point would be a multiple of 2.5 days i.e.
  32 complex points in A-dimension would require an
  80 day experiment
• loss of sensitivity
• an additional transfer step is required
• relaxation takes place during each transfer

Get less data that is less ambiguous?
74
NMR Assignments
4D HNCA

• 4D NMR Experiments
• Backbone Assignments

Correlates 1HCai with NHi NHi1
Correlates NHi with 1HCai 1HCai1
75
NMR Assignments

• 4D NMR Experiments
• Backbone Assignments
• Quality improves with
• deuterium labeling
• TROSY
• specific labeling

J. AM. CHEM. SOC. 9 VOL. 124, NO. 34, 2002