3D Assignments for Web

7/31/2019 3D Assignments for Web

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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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-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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-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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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

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