experiments for backbone and sidechain assignments...
TRANSCRIPT
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.
?
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