Experiments for Backbone and Sidechain Assignments of Uniformly 13 C- and 15 N- Labeled Proteins

Transcription

1 Experiments for Backbone and Sidechain Assignments of Uniformly 13 C- and 15 N- Labeled Proteins

2 Class Outline Understanding 3D experiments Chemical shift assignment experiments Backbone Aliphatic sidechains Aromatic sidechains Stereospecific chemical shifts assignments

3 Resonance Assignment Strategies Depend on the Protein s MW Molecular Weight 8-10 KDa ~ 8-25 KDa 25 KDa Isotopic Labeling None Uniform 13 C, 15 N Uniform 13 C, 15 N, 2 H and/or selective labeling Approach 1 H homonuclear (COSY/TOCSY + NOESY) Triple Resonance 2 H-modified 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.

4 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 15 N and 13 C.

5 Linking Three NMR-active Nuclei Typical 2D experiment showing the correlation between two NMR active nuclei. We could use two 2D experiments: one linking 1 H to 15 N and the other linking 15 N to 13 C. However, it is more efficient to combine these two experiments into one.

6 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.

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

8 Backbone Assignments

9 15 N-HSQC: A Protein s fingerprint? The most common strategy nowadays for backbone chemical shift assignments is to use 15 N-edited 3D experiments.

10 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 protein s backbone.

11 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 isn t recorded, the atom is listed in parentheses. -When possible, avoid duplicating atom names: HN + N + CO will be HNNCO, instead it s abbreviated as HNCO.

12 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 HNCACB Inter- and Intra-residue connections Chemical Shifts HN(i) N(i) C (i-1) C (i-1) HN(i) N(i) C (i-1, i) C (i-1, i)

13 (1) The Assignment Process

14 (2) The Assignment Process -Next we look for a pair of peaks in the CBCA(CO)NH experiment with 13 C 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 ppm.

15 (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 1 H = 8.60 ppm and 15 N = ppm. -Then we continue this way until a brake is found.

16 (4) The Assignment Process

17 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

18 Amino Acids 13 C Chemical Shifts Residue Gly (ppm) Ala Ser Thr Val Leu Ile ( 1) / 14-22( 2) 9-16 Lys Arg Pro Glu Gln Met Phe Tyr His Trp Cys Cys(S-S) Asp Asn Structure of Biological Macromolecules, Rizo and Brunch

19 Chemical shift Info: BMRB (BioMagResBank)

20 Residue Identification

21 More Options for BB Assignments HNCA / HN(CO)CA HNHA / H(CA)NH HNCO / HN(CA)CO

22 Sidechain Assignments

23 Aminoacid Sidechains

24 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 1 H and 13 C sidechain atoms of residue i-1 with the amide 1 H and 15 N of residue i. HN(i) N(i) C (i-1)

25 Sidechain Assignments

26 1 H/ 15 N-TOCSY-HSQC -This experiment allows us to observe intra-residue correlations between the sidechain protons and the amide 1 H/ 15 N.

27 Non HN-based Methods: HCCH- TOCSY -This experiment correlates a 1 H/ 13 C pair to all other protons in the same aminoacid sidechain. -Also, very useful for determining which 1 H is directly attached to which 13 C.

28 Aromatic Sidechains

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

30 Example: Phenylanine a) b) c)

31 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,

32 Stereospecific Assignments

33 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 the 3D 15 N-NOESY experiment to stereospecifically assign them.

34 VAL and LEU Biosynthesis Pro-R Pro-S Pro-R Pro-S

35 CT- 13 C/ 1 H-HSQC Spectra

36 Summary of Assignment Experimets Backbone CBCA(CO)NH, HNCACB, HNCO, HNHA Aliphatics and aromatics sidechains (H)C(CO)NH, H(CCO)NH, HCCH_TOCSY, 15 N-edited- TOCSY, 2D-NOESY, 2D-TOCSY, 2D-COSY, 13 C-HSQC, (H )C (C C )H, and (H )C (C C C )H Stereospecific assignments: -NH 2 (Asn, Gln), Methyls (Val, Leu) 15 N-edited NOESY, CT- 13 C-HSQC

37 Assignment Problem