Jeff Grinstead SB 2006/2007. NMR Spectroscopy. NMR Spectroscopy JG/1 07

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1 NMR Spectroscopy Jeff Grinstead NMR Spectroscopy

2 NMR for structural biology

3 Challenges for determining protein structures using NMR Proteins have thousands of signals Assign the specific signal for each atom Thousands of interactions between atoms also need to be assigned Need to transform the representation from spectra through interactions between atoms to spatial coordinates

4 NMR observables for structure and dynamics Observable chemical shifts 1 H,13C,15N,31P J couplings (through bond) Information assignments, secondary structure 3 dihedral angles: φ, χ, Karplus curves NOE (through space) interatomic distances (<5Å) solvent exchange (HN) hydrogen bonds relaxation / linewidths 1 mobility, dynamics conform./chem. exchange projection angles (ψ, ) residual dipolar couplings projection angles J(HN,Hα), 3J(Hα, Hβ), H,13C,15N H 15N, 1H 13C, 13C 13C, 1

5 Structure determination by NMR Lengthy process: Sample preparation ( months ) Acquisition of experimental data (1 2 months) Chemical shift assignments Backbone (few days) Side chains (few weeks) Analysis of NOE spectra (several months) Structure calculations

6 The resonance assignment puzzle 750 MHz 1H NMR spectrum of the SH3 domain of the tyrosine kinase FYN Hydrogen atoms Ribbon representation

7 Regions of a protein 1H NMR spectrum Side chain CH3 Side chain CH1, CH2 Aromatic ring protons and side chain NH2 Backbone HN Backbone Hα Tryptophan And indole HN ppm

8 Solutions to the Challenges Increase dimensionality of spectra to better resolve signals: Detect signals from heteronuclei (13C,15N) Better resolved signals, different overlaps More information to identify signals Lower sensitivity to MW of protein

9 2D FT NMR: where does the second time dimension come from? The effect of the t1 period is to modulate the amplitude of the signal. By recording successive experiments with increasing t1 periods an amplitude modulation is introduced.

10 2D FT NMR Series of recorded FIDs for increasing t1 periods First Fourier transform in t2 Second Fourier transform in t1

11 2D COSY spectrum: 3 bond scalar couplings Regions in 2D spectra provide protein fingerprints If 2D cross peaks overlap go to 3D

12 Homonuclear Assignment Strategy H strategy: based on backbone NH (unique region of spectrum, greatest dispersion of resonances, least overlap) 1 Scalar coupling to identify spin systems (amino acids), dipolar couplings (NOEs) to place them in sequence Concept: build out from the backbone to determine the side chain resonances 2nd dimension to resolve overlaps, 3D rare

13 Step 1: Identify spin system Intra residue Inter residue

14 Amino acid type Identify resonances for each amino acid Possibilities: Peptide sequence: 1 M/Q/E, A, and L R Q A L M A S

15 Step 2: Link sequential spin systems Based on HN NOEs: A B C or C B A Based on TOCSY, HN Hα NOEs: Intra residue Inter residue A B C, or M/Q/E A L R Q A L M A S

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18 Step 2: Link sequential spin systems Use sequential NOEs to connect amino acids Minor flaw: all NOEs mixed together Use only these to make sequential assignments Tertiary Structure Sequential Intraresidue A B C D Z Medium range (helices)

19 Secondary structure: α helix

20 Hydrogen bonding scheme for an α helix

21 Characteristic NOE patterns

22 Antiparallel β sheet

23 Step 2: Link sequential spin systems Use sequential NOEs to connect amino acids Minor flaw: all NOEs mixed together Use only these to make sequential assignments Tertiary Structure Sequential Intraresidue A B C D Z Medium range (helices)

24 Extended homonuclear 1H strategy Same basic idea as 1H strategy based on backbone HN Concept: when backbone 1H overlaps disperse with backbone 15N 3rd dimension increases signal resolution 1 H < > 1H < > 15N

25 Double resonance experiments increase resolution/information content 15 Correlates proton chemical shifts with chemical shifts of other NMR nuclei such as 15N or 13C (needs labeling!) R N 1H HSQC R 15N Cα CO 15N Cα H H

26 N dispersed 1H 1H TOCSY 15 3 overlapped HN resonances Same HN, different 15N F2 TOCSY HSQC H 1H 1 t1 t2 15 F3 F1 N t3

27 Heteronuclear (1H,13C,15N) strategy Strategy based on backbone 15N 1H (13CO) Concept: when backbone 15N 1H overlaps disperse with backbone C Cα Hα Cβ Hβ 3rd/4th dimension increase signal resolution 3D/4D double/triple resonance NMR 1 > H < > 13C < > 15N < > 1H

28 Large scalar couplings less sensitive to MW of the protein Superior to 1H homonuclear strategy: H H couplings <20 Hz Mixing is faster so less time for signal to relax

29 Sample triple resonance pulse sequence

30 Heteronuclear assignments: backbone experiments HNCA / HN(CO)CA Names of scalar experiments based on atoms detected HN(CA)CO / HNCO HNCACB / HN(CO)CACB Pair of experiments to distinguish between intra and inter residue resonances

31 Heteronuclear assignments: Link amides via matching frequencies HNCA / HN(CO)CA HN (9.6, 113 ppm) HBHA(CBCACO)NH (i 1): CA, HA, CB, HB, CO (i): CA, HA, CB, HB, CO HNCACB / HN(CO)CACB HBHA(CBCA)NH / HN(CA)CO / HNCO

32 HNCO

33 Ambiguity

34 Heteronuclear assignments: Connect sequential amides to protein primary sequence (i 1): CA, HA, CB, HB, CO (i): CA, HA, CB, HB, CO L V S C C Y R S L A A P D L T L R D L E D I V E T S Q A H N A R A Q L T G A L F Y S Q G V F F Q W L E G H P A V A

35 Sample assignment exercise (i 1): CA(59ppm), CB(65ppm) (i): CA(54), CB(29) (i 1): CA(54.7), CB(29.3) (i): CA(45.5), CB(X) (i 1): CA(45.5), CB(X) (i): CA(53.3), CB(33.1) L V S C C Y R S L A A P D L T L R D L E D I V E T S Q A H N A R A Q L T G A L F Y S Q G V F F Q W L E G H P A V A

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37 Sample assignment exercise S Q (i 1): CA(59ppm), CB(65ppm) (i): CA(54), CB(29) G (i 1): CA(54.7), CB(29.3) (i): CA(45.5), CB(X) V (i 1): CA(45.5), CB(X) (i): CA(53.3), CB(33.1) S/T Q/R/C/E/H/M/W/V G V/W/M/K/H/E/C/R L V S C C Y R S L A A P D L T L R D L E D I V E T S Q A H N A R A Q L T G A L F Y S Q G V F F Q W L E G H P A V A

38 Heteronuclear assignments: side chain experiments HBHA(CBCA)NH / HBHA(CBCACO)NH HCCH COSY / HCCH TOCSY

39 Key points about heteronuclear (1H,13C,15N) strategy Most efficient, but experimentally more complex Enables study of large proteins (TROSY) Requires 15N, 13C, [2H] enrichment Target: 1mM protein in 500µL buffer (25kDa protein=12.5mg!) High expression in minimal media (E. coli) ~10 20mg/L good Extra $$ (~$200/g 13C6 glucose, 2g/L media )

40 Practical issues: molecular weight kd for 3D structure > Domains >100 kd: uniform deuteration, residue and site specific, atom specific labeling Symmetry reduces complexity 2 x 10 kd 20 kd TROSY: an experimental approach to higher sensitivity for large systems

41 Transition Next lecture: Practical considerations for NMR Sample applications of NMR to biological problems AppA (a blue light photoreceptor that mediates transcription)

42 NMR Spectroscopy (2) Jeff Grinstead NMR Spectroscopy

43 Ambiguity

44 Sample assignment exercise S Q (i 1): CA(59ppm), CB(65ppm) (i): CA(54), CB(29) G (i 1): CA(54.7), CB(29.3) (i): CA(45.5), CB(X) V (i 1): CA(45.5), CB(X) (i): CA(53.3), CB(33.1) S/T Q/R/C/E/H/M/W/V G V/W/M/K/H/E/C/R L V S C C Y R S L A A P D L T L R D L E D I V E T S Q A H N A R A Q L T G A L F Y S Q G V F F Q W L E G H P A V A

45 Key points about heteronuclear (1H,13C,15N) strategy Most efficient, but experimentally more complex Enables study of large proteins (TROSY) Requires 15N, 13C, [2H] enrichment Target: 1mM protein in 500µL buffer (25kDa protein=12.5mg!) High expression in minimal media (E. coli) ~10 20mg/L good Extra $$ (~$200/g 13C6 glucose, 2g/L media )

46 Practical issues: molecular weight kd for 3D structure > Domains >100 kd: uniform deuteration, residue and site specific, atom specific labeling Symmetry reduces complexity 2 x 10 kd 20 kd TROSY: an experimental approach to higher sensitivity for large systems 50ms T2=500ms (peptide) T2=20ms (big protein)

47 NMR for structural biology purification Protein domain from a database DNA

48 Practical Considerations for Biomolecular NMR Protein size 220aa Solubility 1mM (12.5mg) Stability/aggregation Protein dynamics Deuteration, TROSY bad lineshape, missing peaks $, bugs hate it (Bo, cryoprobe)

49 Problems with higher molecular weights slower tumbling in solution fast decay of NMR signal poor signal to noise larger number of signals overlap in NMR spectra linewidth ν1/2 = 1/πT2 τc MW 5 ns 10 kda 10 ns 20 kda 15 ns 30 kda 25 ns 50 kda

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51 Effect of Cα deuteration in 3D HNCA experiments H no 2H labeling N N HN D HN O H 75 % random fractional 2H labeling HN N N HN O D

52 TROSY and 2H labeling for molecular weights > 50 kda Transverse relaxation optimized spectroscopy H labeling 2 reduced relaxation (γd / γh ~ 1 / 6.5) improved signal to noise better resolution 1 H dipole/dipole relaxation C 13 reduced number of cross peaks suppression of spin diffusion H H H H Pervushin et al. PNAS (1997) 94, D D D D H H N N

53 Practical Considerations for Biomolecular NMR Protein size 220aa Solubility 1mM (12.5mg) Stability/aggregation Protein dynamics Deuteration, TROSY bad lineshape, missing peaks $, bugs hate it (Bo, cryoprobe)

54 Protein domain A folded protein is easily recognized

55 AppA(5 125) Strong:weak peak ratio = 10:1 Flexible termini (long T2) T2(obs) T2+T2(exchange) HSQC screening: Domain boundaries Solution additives Temp, ph Related proteins Related organisms

56 Critical parameters influencing protein production for structure determination by NMR Domain boundaries no soluble expression + 23 aa 4 aa Homologous proteins

57 Multiple Sequence Alignment

58 Practical Considerations for Biomolecular NMR Protein size 220aa Solubility 1mM (12.5mg) Stability/aggregation Protein dynamics Deuteration, TROSY bad lineshape, missing peaks $, bugs hate it (Bo, cryoprobe)

59 Characterizing protein dynamics by NMR: Parameters/timescales µs ms Type of motion Global unfolding Overall tumbling local unfolding Slow loop reorientation Chemical kinetics 10 9 S S flipping Bond librations fast loop reorientation Side chain rotation / reorientation NMR parameter Aromatic ring flips HN exchange Chemical shifts NOE, T2, T1 T2, T1ρ J couplings Dipolar couplings

60 µs ms Timescale, Two Site Exchange

61 Exchange Example: Motion of tyrosine side chains in proteins The environment of tyrosine ring protons is in principle anisotropic in a protein => the various ring protons should give different signals With increasing T ring flips average the 2 6 and 3 5 protons As a result the signals coalesce The interior of proteins is thus NOT rigid! Protein motions play and important role for their function

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63 NMR Applications Protein size 220aa Solubility 1mM (12.5mg) Applications: Stability/aggregation Protein dynamics bad lineshape, missing peaks Deuteration, TROSY $, bugs hate it (Bo, cryoprobe) SAR by NMR H/D exchange lachp O1 complex Transcription factors: transactivator coactivator complexes PYP photoactivation/protein folding AppA photoactivation and transcriptional antirepression via protein interactions

64 Rational drug design: SAR by NMR Structure-Activity Relationships by NMR Science (1996) 274, 1531 SAR by NMR is a nuclear magnetic resonance (NMR) based method in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high affinity ligands. The approach is called "SAR by NMR" because structure activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for a target protein can be rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high affinity ligands and is particularly useful in target directed drug research.

65 NMR Applications Protein size 220aa Solubility 1mM (12.5mg) Applications: Stability/aggregation Protein dynamics bad lineshape, missing peaks Deuteration, TROSY $, bugs hate it (Bo, cryoprobe) SAR by NMR H/D exchange lachp O1 complex Transcription factors: transactivator coactivator complexes PYP photoactivation/protein folding AppA photoactivation and transcriptional antirepression via protein interactions

66 NMR studies of protein folding thermodynamics/kinetics N H (closed) kop N H (open) kcl S31 kop kch D2O N D Q55 Opening rates (h 1) EX1 [kobs=kop] 0.20 EX2 k ex =(k op / k cl ) k ch =P kch kch (s 1) ph free Backbone amide protection factors of lac HP62 DNA bound QuickTime and a Animation decompressor are needed to see this picture. Kalodimos et al. Nature Struct. Biol. 2002, 9, 193. Kalodimos et al Nature Struct Biol 9, 193

67 NMR Applications Protein size 220aa Solubility 1mM (12.5mg) Applications: Stability/aggregation Protein dynamics bad lineshape, missing peaks Deuteration, TROSY $, bugs hate it (Bo, cryoprobe) SAR by NMR H/D exchange lachp O1 complex Transcription factors: transactivator coactivator complexes PYP photoactivation/protein folding AppA photoactivation and transcriptional antirepression via protein interactions

68 Transcription RNA Polymerase II

69 Transcriptional regulation CREB P competitive inhibition (340aa) gene repression KIX domain (80aa) Of CBP (2400aa)

70 A folded protein is easily recognized Coactivator bound TF

71 NMR Applications Protein size 220aa Solubility 1mM (12.5mg) Applications: Stability/aggregation Protein dynamics bad lineshape, missing peaks Deuteration, TROSY $, bugs hate it (Bo, cryoprobe) SAR by NMR H/D exchange lachp O1 complex Transcription factors: transactivator coactivator complexes PYP photoactivation/protein folding AppA photoactivation and transcriptional antirepression via protein interactions

72 NMR studies of protein folding photoactive yellow protein pg Ectothiorhodospira halophila pg light pb E46Q pr sub s sub ms pb Rubinstenn et al 1998, Nature Struct Biol 7, 568

73 Laser illumination glass fiber Shigemi tube laser

74 pb state of wt PYP Disappearing peaks: µs ms timescale exchange Protein unfolds Blue: peaks in pb disappear Red: peaks in pb still visible Nocky Derix

75 NMR Applications Protein size 220aa Solubility 1mM (12.5mg) Applications: Stability/aggregation Protein dynamics bad lineshape, missing peaks Deuteration, TROSY $, bugs hate it (Bo, cryoprobe) SAR by NMR H/D exchange lachp O1 complex Transcription factors: transactivator coactivator complexes PYP photoactivation/protein folding AppA photoactivation and transcriptional antirepression via protein interactions

76 A new photoreceptor: AppA Rhodobacter sphaeroides Photosynthesis genes Masuda, S. (2002) Cell 110, 613.