Experiments for Backbone and Sidechain Assignments of Uniformly 13C- and 15NLabeled 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 Proteins MW

Molecular Weight 8-10 KDa ~ 8 - 25 KDa 25 KDa Isotopic Labeling
1H

Approach homonuclear (COSY/TOCSY + NOESY) Triple Resonance

2H-modified

None Uniform 13C, 15N Uniform 13C, 15N, 2H and/or selective labeling

triple resonance

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.

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 be isotopically labeled with 15N and 13C.

Linking Three NMR-active Nuclei

Typical 2D experiment showing the correlation between two NMR active nuclei.

We could use two 2D experiments: one linking 1H to 15N and the other linking 15N to 13C. However, it is more efficient to combine these two experiments into one.

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 nuclei around the peptide bond.

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

Backbone Assignments

15N-HSQC:

A Proteins fingerprint

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

The Protein Backbone

3D NMR experiments for chemical shift assignments are based on the ability to transfer magnetization through NMR active nuclei using J couplings. Many of these experiments have been designed to walk through the proteins backbone.

3D NMR Methods: 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.

3D NMR Methods: CBCA(CO)NH and HNCACB

-These experiments are the workhorse for backbone assignments. -Both provide similar sets of data: CBCA(CO)NH
Inter-residue connections only

Chemical Shifts
HN(i) N(i) C (i-1) C (i-1)

HNCACB
Inter- and Intra-residue connections

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

(1) The Assignment Process

(2) The Assignment Process

-Next we look for a pair of peaks in the CBCA(CO)NH experiment with 13C 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.

(3) The Assignment Process

-To obtain the chemical shifts of C (i+1) and C (i+1) we have to find the HNCACB strip corresponding to 1H = 8.60 ppm and 15N = 117.0 ppm. -Then we continue this way until a brake is found.

(4) The Assignment Process

Primary Sequence Identification

-Until now we only know the order in which our anonymous spin systems (HN/N/C /C ) are arranged. 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

Amino Acids
Residue
Gly Ala Ser Thr Val Leu Ile Lys Arg Pro Glu Gln Met Phe Tyr His Trp Cys Cys(S-S) Asp Asn 42-48 (ppm) 49-56 55-62 58-68 57-67 51-60 55-66 52-61 50-60 60-67 52-62 52-60 51-59 52-64 54-63 53-60 55-63 53-59 54-61 50-58 49-57

13C
18-24 61-67 66-73 30-37 39-48 34-47 29-37 28-35 27-35 27-34 24-33 30-38 36-44 37-45 27-36 28-29 29-33 40-48 38-45 33-41

Chemical Shifts

19-26 16-25 22-29 25-31( 1) / 14-22( 2) 21-26 25-30 24-29 32-38 32-36 31-35 21-28 9-16 27-34 41-45 49-53 40-43

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

-A variety of sidechaindirected experiments are available to identify sidechain chemical shifts. For example: H(CCO)NH and (H)C(CO)NH.

Chemical Shifts
HN(i) N(i) H (i-1)

-These experiments correlate the 1H and 13C sidechain atoms of residue i-1 with the amide 1H and 15N of residue i.

HN(i) N(i) C (i-1)

Sidechain Assignments

1H/15N-TOCSY-HSQC
-This experiment allows us to observe intra-residue correlations between the sidechain protons and the amide 1H/15N.

Non HN-based Methods: HCCHTOCSY

-This experiment correlates a 1H/13C pair to all other protons in the same aminoacid sidechain. -Also, very useful for determining which 1H is directly attached to which 13C.

Aromatic Sidechains

Assignment Strategy #1
a) b) c) Link protons with aromatic ring protons: 2D-NOESY in D2O Assign ring protons: 2D-COSY, 2D-TOCSY in D2O Assign aromatic carbons: 1H/13CHSQC

Example: Phenylanine
a)

b)

c)

Assignment Strategy #2
It could be difficult to obtain complete assignments of aromatic residues when the aromatic protons have a high density of NOEs and poor chemical shift dispersion. Another strategy consists in correlations between the sidechain C 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 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 the 3D 15N-NOESY experiment to stereospecifically assign them.

VAL and LEU Biosynthesis

Pro-R Pro-S

Pro-R Pro-S

CT-13C/1H-HSQC Spectra

Summary of Assignment Experimets

Backbone
CBCA(CO)NH, HNCACB, HNCO, HNHA

Aliphatics and aromatics sidechains

(H)C(CO)NH, H(CCO)NH, HCCH_TOCSY , 15N-editedTOCSY, 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