Experiments for Backbone

and Sidechain Assignments

of Uniformly 13C- and 15N-

Labeled Proteins

Carlos A. Amezcua

April 18, 2006

Class Outline

Understanding 3D experiments

Chemical shift assignment experiments

Backbone

Aliphatic sidechains

Aromatic sidechains

Stereospecific chemical shifts assignments

Resonance Assignment Strategies

Depend on the Protein’s MW

2H-modified triple

resonance

Uniform 13C, 15N, 2H

and/or selective

labeling

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 incrowded and overlapped spectra. Additionally, the proteins tumble slower insolution which results in broader peaks.

What do we Mean by

Resonance Assignment?

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 beisotopically labeled with 15N and 13C.

Linking Three NMR-active Nuclei

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 thecorrelation between two NMR activenuclei.

3D NMR Experiments

Each peak has 3 chemical shifts associated with it: HN, N, and CO

In this example each peak contains information about the NMR active nucleiaround the peptide bond.

Analysing 3D Data

We can take 2D slices from the 3D cube alongthe 15N dimension and associate HN with CO.

Backbone

Assignments

15N-HSQC: A Protein’s fingerprint

The most common strategy nowadays for backbone chemicalshift assignments is to use 15N-edited 3D experiments.

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The Protein Backbone

3D NMR experiments for chemical shift assignments are based on the abilityto transfer magnetization through NMR active nuclei using J couplings.

Many of these experiments have been designed to “walk” through theprotein’s backbone.

3D NMR Methods: HNCO

Recorded chemical shifts:

HN (i), N (i), CO (i-1)

Nomenclature:

-The experiment name lists the atomswhose chemical shifts are recorded.

-When the magnetization transfer isthrough nuclei whose chemical shiftisn’t recorded, the atom is listed in“parentheses”.

-When possible, avoid duplicatingatom names: HN + N + CO will be“HNNCO”, instead it’s abbreviated as“HNCO”.

3D NMR Methods: CBCA(CO)NH

and HNCACB

Chemical ShiftsHN(i)N(i)

C (i-1)C (i-1)

HN(i)N(i)

C (i-1, i)C (i-1, i)

-These experiments arethe workhorse forbackbone assignments.

-Both provide similar setsof data:

CBCA(CO)NH

Inter-residue connectionsonly

HNCACB

Inter- and Intra-residueconnections

(1) The Assignment Process

(2) The Assignment Process

-Next we look for a pair of peaks inthe CBCA(CO)NH experiment with13C chemical shifts of 30 and 57.5ppm. This strip gives us the chemicalshift of HN (i+1) and N (i+1) : 8.60and 117.0 ppm.

(3) The Assignment Process

-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 isfound.

(4) The Assignment Process

Primary Sequence Identification

-Until now we only know the order in which ouranonymous spin systems (HN/N/C /C ) arearranged. However, we want to know the amino acidtype to which each belongs.

-We start by identifying those spin systems thathave 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

Amino Acids 13C Chemical Shifts

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

Chemical shift Info: BMRB

(BioMagResBank)

www.bmrb.wisc.edu

Residue Identification

More Options for BB Assignments

HNCA / HN(CO)CA HNHA / H(CA)NH HNCO / HN(CA)CO

Sidechain

Assignments

Aminoacid Sidechains

SC Assignment by 3D-NMR:

H(CCO)NH- and (H)C(CO)NH-TOCSY

Chemical ShiftsHN(i)N(i)

H (i-1)

HN(i)N(i)

C (i-1)

-A variety of sidechain-directed experiments areavailable to identifysidechain chemical shifts.For example: H(CCO)NHand (H)C(CO)NH.

-These experimentscorrelate the 1H and 13Csidechain atoms of residuei-1 with the amide 1H and15N of residue i.

Sidechain Assignments

1H/15N-TOCSY-HSQC

-This experiment allows usto observe intra-residuecorrelations between thesidechain protons and theamide 1H/15N.

Non HN-based Methods: HCCH-

TOCSY

-This experiment correlates a1H/13C pair to all other protonsin the same aminoacidsidechain.

-Also, very useful fordetermining which 1H isdirectly attached to which 13C.

Aromatic

Sidechains

Assignment Strategy #1

a) Link protons with aromaticring protons: 2D-NOESY in D2O

b) Assign ring protons: 2D-COSY,2D-TOCSY in D2O

c) Assign aromatic carbons: 1H/13C-HSQC

Example: Phenylanine

a)

b) c)

Assignment Strategy #2

It could be difficult to obtaincomplete assignments of aromaticresidues when the aromaticprotons have a high density ofNOEs and poor chemical shiftdispersion.

Another strategy consists incorrelations between the sidechainC and ring H / chemical shifts

using J-couplings.

Experiment names:(H )C (C C )H and(H )C (C C C )H

Yamazaki, T., Forman-Kay, J.D, and Kay, L.E, JACS (1993), 115, 11054-11055

Stereospecific

Assignments

ASN and GLN NH2 Groups

•The sidechain CO-N bond haspartial double bond character.

•Rotation around this bond isslow in the NMR time scale.

•The distance between the Eproton and the (Asn) or (Glu) protons is smaller thanfor the Z proton.

•Use relative intensities in the3D 15N-NOESY experiment tostereospecifically assign them.

VAL and LEU Biosynthesis

Pro-R Pro-R

Pro-S Pro-S

CT-13C/1H-HSQC Spectra

Summary of Assignment

Experimets

BackboneCBCA(CO)NH, HNCACB, HNCO, HNHA

Aliphatics and aromatics sidechains(H)C(CO)NH, H(CCO)NH, HCCH_TOCSY , 15N-edited-

TOCSY, 2D-NOESY, 2D-TOCSY, 2D-COSY, 13C-HSQC,

(H )C (C C )H , and (H )C (C C C )H

Stereospecific assignments: -NH2 (Asn, Gln),Methyls (Val, Leu)

15N-edited NOESY, CT-13C-HSQC

Assignment Problem