Principles of NMR Protein Spectroscopy. 2) Assignment of chemical shifts in a protein ( 1 H, 13 C, 15 N) 3) Three dimensional structure determination

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1 1) Protein preparation (>50 aa) 2) Assignment of chemical shifts in a protein ( 1 H, 13 C, 15 N) 3) Three dimensional structure determination

2 Protein Expression overexpression in E. coli - BL21(DE3) 1 H only (unlabelled) - LB or other rich source 15 N-specific label - 15 N aa, auxotroph E. coli strain 1 H, 13 C, 15 N (labelled) - M9 minimal 13 C-glucose - carbon source 15 N-NH 4 Cl - nitrogen source 2 H, 13 C, 15 N (labelled) - deuterated proteins 2 H, 13 C-glucose, 2 H 2 O

3 Protein Expression - other variations Gardner & Kay (1998) Ann. Rev. Biophys. Biomol. Str. 27, Goto & Kay (2000) Curr. Opin. Str. Biol. 10,

4 Sample Preparation mm protein (10 mg for 20 kda protein) ml buffer (CH 3 COONa, NaH 2 PO 4, HEPES etc) mm NaCl, KCl or other ph 2-8 H 2 O or H 2 O/D 2 O 5 C - 60 C

5 How do we Assign a Protein ie. determine all 1 H, 15 N and 13 C chemical shifts 1-5 aa - use 1D NMR - based on chemical shift tables aa - use 2D NMR - correlate 2 chemical shifts 1 H- 1 H, 1 H- 15 N, 1 H- 13 C >100 aa - use 3D NMR - correlate 3 chemical shifts 1 H- 1 H- 13 C, 1 H- 1 H- 15 N, 1 H- 13 C- 15 N

6 Protein is 13 C, 15 N labelled correlate 15 N shifts with attached 1 H (or 13 C- 1 H) D-R-E-V-I-L-I-S-H-I-P 1 H spectrum 15 N spectrum ppm ppm

7 Chemical Shift Assignment 1 H- 15 N Shift Correlation (HMQC or HSQC) D-R-E-V-I-L-I-S-H-I-P ppm H N O N H O ppm a fingerprint spectrum of the protein

8 1 H- 15 N Shift Correlation (HMQC) 1 H 1/2J 1/2J 15 N t 1 Decouple HN J=93 Hz HC J=143 Hz 5.37 msec 3.50 msec

9 Protein Expression - other variations Gardner & Kay (1998) Ann. Rev. Biophys. Biomol. Str. 27,

10 Chemical Shift Assignment. But how do we get the assignments D-R-E-V-I-L-I-S-H-I-P.. correlate three nuclei from different residues HNCACB - correlates HN with N and Cα, Cβ shifts (i, i-1) CBCA(CO)NH - correlates HN with N and i-1 Cα, Cβ shifts E V I L H N O N H O H N O N H O

11 Chemical Shift Assignment HNCACB, CBCA(CO)NH are 3D NMR methods ppm ppm C ppm 1 H ppm 15 N

12 CBCA(CO)NH, HNCACB Strategy HNCACB Strips Kanelis et al (2001)

13 Chemical Shift Assignment HNCA HN(CO)CA HNCO H(CCO)NH C(CO)NH HCCH-TOCSY

14 Table of chemical shift assignments Retrieve

15 Table of chemical shift assignments - BMRB other information

16 Structure Determination by NMR Spectroscopy 1) Sequence of protein 2) Table of chemical shift assignments 3) Secondary structure - Chemical Shift Index 4) Three Dimensional Structure -we need info about how structure is folded -use 1 H- 1 H distances -will use existing NMR assignments and simply give correlations between 1 Hs close in space

17 Chemical Shift Index Recall ν s = γb 0 2π σ γb 0 2π (Hz) σ = shielding constant φ,ψ affect this also α-helix β-sheet φ ~ -60, ψ ~ 40 φ ~ -140, ψ ~ 140 Chemical shifts of backbone atoms should depend on α-helix or β-sheet structure

18 Chemical Shift Index Wishart et. al. (1992) Biochemistry 31, α-helix β-sheet Ηα δ < average ± range δ > average ± range Cβ δ < average ± range δ > average ± range C δ > average ± range δ < average ± range Cα δ > average ± range δ < average ± range ppm 1 0 ppm

19 Chemical Shift Index _Residue_label _Atom_name _Atom_type _All_chem_shift_value _Coil_chem_shift_value _Beta_strand_chem_shift_value _Helix_chem_shift_value _CSI_chem_shift_value _CSI_chem_shift_range ALA H H ALA HA H ALA C C ALA CA C ALA CB C ALA N N

20 Chemical Shift Index Value Hα, Cβ δ < average ± range -1.0 (helix) δ > average ± range 1.0 (sheet) Cα, C δ > average ± range -1.0 (helix) δ < average ± range 1.0 (sheet) Examine Values for each residue (max. 4) > 2/3 or 3/4 for each, define residue as 1 or -1 4 or more -1s α-helix 3 or more 1s β-sheet

21 Chemical Shift Index - calculate from chemical shift tables β-sheet α-helix β-sheet α-helix α-helix - provides 2 structure - use 1 H- 1 H distances to determine entire structure

22 Structure Determination Nuclear Overhauser Effect (noe) H i r ij H j noe η ij = γ 4 h 2 (τ app - 6τ app ) 10r ij ωτ app

23 Structure Determination Nuclear Overhauser Effect (noe) H i r ij H j noe (η ij ) - change in intensity of an NMR resonance when the transitions of another are perturbed η ij = (Ι Ι 0 ) I 0 η Ηα = Κ r Ηα 6 N H Hε Hδ Hα Hζ Hβ Hβ O Hε Hδ 2.46 Å η δε = Κ r δε 6 η Ηα = η δε r δε 6 r Hα 6

24 13 C or 15 N NOESY Kanelis et al (2001)

25 Structure Determination 1) Sequence of protein 2) Table of chemical shift assignments 3) Secondary structure - CSI 4) Distance information from noe Calculate Structure

26 Structure Determination What information do we have 1) Polypeptide chain - sequence, atoms, bonds 2) Distances between 1 H- 1 H pairs 3) φ,ψ angles from CSI (TALOS) using chemical shifts INPUT computer algorithm that attempts to satisfy all Xplor, CNS, DIANA, GROMOS Repeat x - look for similar fold unique structure

27 Structure Determination NMR Data Random Structure Bonding Info Distance / Torsional Restraints Folding E noe = k(r c -r u ) 2 E dih = k(φ c -φ u ) 2 Global Fold Refinement H-bonds RDC Final Structures

28 Structure Determination What to look for. 1) Many noes > 10 / residue, 200aa >2000 distances 2) Representative family of structures ~ 20 well structured bb rms ~ 0.5 ± 0.1 Å 3) Ramachandran Plot - > 85% most favoured 4) Poorly defined areas - higher rms may be flexible or lack of noes 5) Inconsistancies in distances r obs r calc no violations > 0.3 Å 6) Structures deposited + NMR data