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Ex perim ent s fo r Bac k boneand Sidec hain Ass ignm ent s
of Un i form ly 13C- and 15N-
Labeled Prot e ins
Carlos A. Amezcua
April 18, 2006
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Class Out l ine
Understanding 3D experiments Chemical shift assignment experiments
Backbone
Al ipha t i c s idecha ins
Aromat ic s idecha ins
Stereospecific chemical shifts assignments
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Resonanc e Ass ignm ent St ra t eg ies
Depend on t he Prot e in s MW
2H-modified triple
resonance
Uniform 13C, 15N, 2H
and/or selectivelabeling
25 KDa
Triple ResonanceUniform 13C, 15N~ 8 - 25 KDa
1H homonuclear
(COSY/TOCSY +
NOESY)
None 8-10 KDa
ApproachIsotopic LabelingMolecular Weight
As the molecular weight increases the number of peaks also increase, resulting in
crowded and overlapped spectra. Additionally, the proteins tumble slower in
solution which results in broader peaks.
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What do w e Mean by
Resonanc e Assignm ent ?
First we make a list of the
NMR active nuclei in the
protein. Second, we identify their
chemical shifts from
NMR spectra.Note: In this example the protein should be
isotopically labeled with 15N and 13C.
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Link ing Three NMR-ac t ive Nuc le i
We could use two 2D experiments: one linking1H to 15N and the other linking 15N to 13C.
However, it is more efficient to combine thesetwo experiments into one.
Typical 2D experiment showing the
correlation between two NMR active
nuclei.
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3D NMR Ex per im ent s
Each peak has 3 chemical shifts associated with it: HN, N, and CO
In this example each peak contains information about the NMR active nuclei
around the peptide bond.
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Analys ing 3D Dat a
We can take 2D slices from the 3D cube along
the 15N dimension and associate HN with CO.
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Backbone
Ass ignments
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15N-HSQC: A Pro t eins f ingerp r int
The most common strategy nowadays for backbone chemical
shift assignments is to use 15N-edited 3D experiments.
?
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The Pro t e in Bac k bone
3D NMR experiments for chemical shift assignments are based on the ability
to transfer magnetization through NMR active nuclei using Jcouplings.
Many of these experiments have been designed to walk through the
proteins backbone.
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3D NMR Met hods: HNCO
Recorded chemical shifts:
HN (i), N (i), CO (i-1)
Nomenclature:
-The experiment name lists the atoms
whose chemical shifts are recorded.
-When the magnetization transfer is
through nuclei whose chemical shift
isnt recorded, the atom is listed in
parentheses.
-When possible, avoid duplicating
atom names: HN + N + CO will be
HNNCO, instead its abbreviated as
HNCO.
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3D NMR Met hods : CBCA(CO)NH
and HNCACB
Chemical Shifts
HN(i)
N(i)
C(i-1)
C(i-1)
HN(i)
N(i)
C(i-1, i)
C(i-1, i)
-These experiments are
the workhorse for
backbone assignments.
-Both provide similar sets
of data:
CBCA(CO)NH
Inter-residue connections
only
HNCACB
Inter- and Intra-residue
connections
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(1) The Assignm ent Proc ess
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(2) The Assignm ent Proc ess
-Next we look for a pair of peaks in
the CBCA(CO)NH experiment with13C chemical shifts of 30 and 57.5
ppm. This strip gives us the chemical
shift of HN (i+1) and N (i+1) : 8.60
and 117.0 ppm.
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(3) The Assignm ent Proc ess
-To obtain the chemical shifts of C(i+1)and C(i+1) we have to find the HNCACBstrip corresponding to 1H = 8.60 ppm and15N = 117.0 ppm.
-Then we continue this way until a brake is
found.
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(4) The Assignm ent Proc ess
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Pr im ary Sequenc e Ident i f i c a t ion-Until now we only know the order in which our
anonymous spin systems (HN/N/C/C) arearranged. However, we want to know the amino acid
type to which each belongs.
-We start by identifying those spin systems that
have unique chemical shifts. For example:
Gly: No C and C ~ 45ppm
Ser/Thr: C is downfield of C (~65-75ppm)
Ala: C is particularly upfield (~15-20ppm)
J. Cavanagh, et al., Protein NMR Spectroscopy, 1996
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Am ino Ac ids
13
C Chem ic a l Shi f t s
9-1625-31( 1) / 14-22( 2)34-4755-66Ile
33-4149-57Asn
38-4550-58Asp
40-4854-61Cys(S-S)
29-3353-59Cys
28-2955-63Trp
27-3653-60His
37-4554-63Tyr
36-4452-64Phe
31-3530-3851-59Met
32-3624-3352-60Gln
32-3827-3452-62Glu
49-5324-2927-3560-67Pro
41-4525-3028-3550-60Arg
40-4327-3421-2629-3752-61Lys
21-2822-2939-4851-60Leu
16-2530-3757-67Val
19-2666-7358-68Thr
61-6755-62Ser
18-2449-56Ala
42-48 (ppm)Gly
Residue
Structure of Biological Macromolecules, Rizo and Brunch
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Chem ic al sh i f t In fo: BMRB(BioMagResBank)
www.bmrb.wisc.edu
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Residue Ident i f ic a t ion
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More Opt ions for BB Ass ignm ent s
HNCA / HN(CO)CA HNHA / H(CA)NH HNCO / HN(CA)CO
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Sidechain
Ass ignments
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Am inoac id Sidec ha ins
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SC Ass ignm ent by 3D-NMR:
H(CCO)NH- and (H)C(CO)NH-TOCSY
Chemical Shifts
HN(i)
N(i)
H(i-1)
HN(i)
N(i)
C(i-1)
-A variety of sidechain-
directed experiments are
available to identify
sidechain chemical shifts.
For example: H(CCO)NHand (H)C(CO)NH.
-These experiments
correlate the 1H and 13C
sidechain atoms of residuei-1 with the amide 1H and15N of residue i.
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Sidec ha in Ass ignm ent s
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1H/15N-TOCSY-HSQC
-This experiment allows us
to observe intra-residue
correlations between the
sidechain protons and the
amide 1H/15N.
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Non HN-based Met hods: HCCH-
TOCSY
-This experiment correlates a1H/13C pair to all other protons
in the same aminoacid
sidechain.
-Also, very useful for
determining which 1H is
directly attached to which 13C.
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Aromat i c
Sidechains
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Ass ignm ent St ra t egy #1
a) Link protons with aromatic
ring protons: 2D-NOESY in D2O
b) Assign ring protons: 2D-COSY,
2D-TOCSY in D2O
c) Assign aromatic carbons: 1H/13C-
HSQC
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Ex am ple: Phenylanine
a)
b) c)
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Ass ignm ent St ra t egy #2
It could be difficult to obtain
complete assignments of aromaticresidues when the aromaticprotons have a high density ofNOEs and poor chemical shiftdispersion.
Another strategy consists in
correlations between the sidechainC and ring H/ chemical shiftsusing J-couplings.
Experiment names:(H)C(CC)H and(H)C(CCC)H
Yamazaki, T., Forman-Kay, J.D, and Kay, L.E, JACS (1993), 115, 11054-11055
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Stereospec i f ic
Ass ignments
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ASN and GLN NH 2 Groups
The sidechain CO-N bond has
partial double bond character.
Rotation around this bond is
slow in the NMR time scale.
The distance between the E
proton and the (Asn) or
(Glu) protons is smaller than
for the Z proton.
Use relative intensities in the3D 15N-NOESY experiment to
stereospecifically assign them.
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VAL and LEU Biosynt hes is
Pro-R Pro-R
Pro-S Pro-S
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CT-13C/1H-HSQC Spec t ra
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Sum m ary o f Assignm entExper imets
Backbone CBCA(CO)NH, HNCACB, HNCO, HNHA
Aliphatics and aromatics sidechains
(H)C(CO)NH, H(CCO)NH, HCCH_TOCSY , 15N-edi ted-TOCSY, 2D-NOESY, 2D-TOCSY, 2D-COSY, 13C-HSQC,
(H)C(CC)H, and (H)C(CCC)H
Stereospecific assignments: -NH2 (Asn, Gln),
Methyls (Val, Leu)
15N-ed it ed NOESY, CT-13C-HSQC
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Assignm ent Prob lem