Where do we stand on Projection NMR Spectroscopy? Thomas Szyperski Chianti Workshop 06/05/07

Transcription

1 Where do we stand on Projection NMR Spectroscopy?

2 Definition: Projection Mapping an N dimensional vector space onto an N-K dimensional sub-space Associated Definitions Specify field over which vector space is defined Dimensionality N of vector space Dimensionality N-K of sub-space Mapping associated with projection Retrieval of N dimensional information

3 Definition: GFT Projection NMR (early 2002) Mapping an N > 2 dimensional conventional spectrum onto 2 K N t 2 dimensional projected spectra Associated Definitions Vector space: complex Dimensionality of conventional spectrum: N arbitrary Dimensionality of projected spectra: N > N t 2 Mapping associated with projection: linear combinations of shifts measured phase-sensitively sensitively in pure absorption mode Retrieval of N dimensional spectral information: Unambiguous identification of chemical shift multiplets: Central peak detection ( orthogonal( projections ) ) and/or differential scaling of jointly ( radially( radially ) ) sampled shift evolution periods Calculation of shifts from a system of equations derived from the e linear combinations of chemical shifts Validation of retrieval of N dimensional spectral information

4 4D FT NMR spectrum (Ω 1, Ω 2, Ω 3, Ω 4 ) ω 1 ω 2 ω 3 ω 4

5 (4,2)D GFT NMR experiment Ω 1 + Ω 2 + Ω 3 Ω 1 + Ω 2 - Ω 3 Ω 1 - Ω 2 + Ω 3 Ω 1 - Ω 2 - Ω 3 ω 1 (GFT) Ω 1 + Ω 2 Ω 1 - Ω 2 measurement of linear combinations of shifts in different sub-spectra spectra phase-sensitivelysensitively and in pure absorption mode challenge: unambiguous identification of shift multiplets Ω 1 ω 2

6 Key challenge: Phase sensitive - Pure absorption mode Projected spectra (4,2)D GFT NMR

7 In general:

8 Validation: Dimensionality of N D spectrum was retrieved intensities scaling central peaks

9

10

11 Definition: 3D->2D Projection in space Mapping an N = 3 dimensional object onto an 2 dimensional projection plane Associated Definitions Vector space: real Dimensionality of object: 3 Dimensionality of projection: 2 Mapping associated with projection: e.g., integral along projection direction Retrieval of 3D information Reconstruction (e.g., Radon Transformation)

12 Methodology Transfer for NMR

13 Projection Reconstruction (PR) NMR (Kupce & Freeman, JACS 2004, 126, 6429)

14 Projection component in Projection-reconstruction reconstruction (PR) NMR is equivalent to GFT projection NMR

15 (4,2)D GFT NMR

16 G-matrix transformation standard hyper-complex FT

17 Scaling of shift evolution periods tilt angles of plane projection

18

19

20 tilted projected spectrum

21 Role of G-matrix G transformation in Projection NMR spectroscopy Phase-sensitive sensitive detection of linear of combinations of chemical shifts combined with (time domain) editing into pure absorption mode sub-spectra spectra

22 Major developments in the s 1980s (Richard Ernst and coworkers) Skewed projections of homo-nuclear 2D J-spectra were calculated using the projection cross-section section theorem to obtain decoupled 1D 1 H NMR spectra; No o joint incrementation of shift evolution periods or phase- sensitive signal detection No pure absorption mode spectra Accordion NMR : joint sampling of chemical shift evolution period and mixing time in EXSY -> Reduction in dimensionality

23

24 The Projection cross section theorem is a blunt weapon in Projection NMR: was derived for N to N-1 N 1 dimensional projections (Bracewell( Bracewell,, 1956); does not imply how to edit components of shift multiplets into sub-spectra; spectra; does not imply how to implement phase sensitive detection; does not imply how to obtain pure absorption mode sub-spectra; spectra;

25

26 A major challenge for future development of Projection NMR: Demand for a generally accepted nomenclature

27

28

29

30

31

32

33 Classification of Projected NMR spectra Which chemical shifts are jointly sampled? Dimensionality of the spectral information? Are the projected sub-spectra spectra used to reconstruct conventional ND spectra?

34 GFT Projection NMR records

35 Pilot study (3.2004): Structure of 14 kda YqfB 16.9 hrs GFT spectra for resonance assignment and 9.1 hrs for simultaneous 3D NOESY (1 mmol solution at 25 o C and 600 MHz w/cryogenic probe)

36

37 Highest-dimensional spectral Information for a protein: 6D H αβ C αβ C α CONHN in 29 hours

38 Expansion of the GFT NMR arsenal..

39 Longitudinal relaxation optimized GFT NMR: L-GFT SoFast,, Best NMR Ultrasofast NMR: Frydman, Brutscher et al.

40 L-GFT NMR for aromatic rings

41 Ring flipping and functional dynamics in the 21 kda protein HR41

42 (4,3)D [HN/H 13 C ali ]-NOESY-[NH/ 13 H]: 13 C ali H]: Rapid acquisition of highly resolved 4D NOESY information

43 NOEs directly assigned in 3D NOESY based on shifts only: without with (4,3)D NOESY

44 G 2 FT NMR: a contribution to establish NMR-based structural genomics of membrane proteins and to study (partially) unfolded proteins

45 (5,3)D HN{N,CO CO}{ }{C αβ C α }

46 GFT NMR applied for structural genomics

47 Approaches for rapid NMR data collection

48 PSI-1: NMR based structural genomics works and is often the only choice

49 NESGC NMR Program Five experimental research groups 15 FTE Ph.D. level researchers 88 NMR structures in PSI-2 (84 in PSI-1) Strong methodology development component Publications in PSI-2: 41

50 NESG NMR Structures from Szyperski Lab: MW distribution MW [Da] ER75 TT212 QR6 MR19 GR2 PfR13 flua CcR19 ET99 HR532 VT1 PfR14 MaR11 SR215 ET95 BcR68 BhR29 XcR50 MrR16 PaT85 HR41 SR220 HR2106 NeT3 ER226 HdR14 SoR39 StR70 PaT89 SR482 ER415 SR213 PaT90 SR355 TT821A GR101 Str106 GR83 StR109 CgR3 CgR1 SsR105 Sr500a SsR10 rpt8 MR32 BcR54 VcR36 NESG ID

51 OR9 - tetramer 2g9j MW 16,108 MONTELIONE HR dimer 2b MW 21,844 SZYPERSKI SR450 - dimer 2dsm MW 14,152 KENNEDY StR109 - dimer 2jna MW 20,606 SZYPERSKI OR1 - dimer 1ihg MW 8,646 MONTELIONE GR83 - dimer 2nwt MW 14,152 SZYPERSKI

52 Protein yjbr from E. coli -ER226 Li N, Sickmier E A, Zhang R, Joachimiak A, White S W. Mol. Microbiol 2002; III 43:1079. N I B F A E C D C II IV C I I C B A N C N B A III C D F F IV E II E III D II PF04237 with 122 proteins without functional assignment Five β-strands A( ), B( ), C( ), D( ), F( ) and E( ) Dali: C-terminal domain (1kaf) of the bacteriophage T4 transcription factor MotA Recently discovered DNAbinding motif : "double wings" Sequence conservation within Pfam04237: all 122 members are DNA binding domains with double wing motif High leverage value Singarapu, Liu, Xiao, Bertonati, Honig, Montelione, Szyperski (2007) Proteins 65,

53 J-GFT NMR for Precise Measurement of Mutually Correlated Nuclear Spin-spin Couplings Residual Dipolar Couplings (RDCs): orientational constraints Structure validation and refinement (Homo-dimers, e.g. GR83) Determination of relative domain orientation Fold determination with sparse NOE networks Support of resonance assignment Identification of secondary structure Investigation of protein dynamics Complement of in silico structure prediction Simultaneous, correlated measurement of different types of RDCs Single NMR experiment: no variation of r.f. duty cycles Resolve chemical shift degeneracy RDCs are grouped according to spin system even if no sequential resonance assignments are available

54 J-GFT (6,2)D (HA-CA-CO)-N-HN K = D + J K C α H α H α C α K C α C C K C N H N N K NH N

55 J-GFT NMR: concept Spin-spin couplings are real numbers Chemical shifts are represented by complex numbers pseudo phase sensitive detection: cos(ωt) ) + i sin(ωt) J-GFT requires detection of cos(πκt) + i sin(πκt) no additional evolution periods Extension of GFT NMR formalism / new approach for sampling J-evolution in time domain

56

57

58 Phase correction of real component only

59

60 8 KDa protein Z-domain non-aligned (blue), aligned with Pf1 phages (red)

61 Summary J-GFT NMR Approach for simultaneous, correlated and precise measurement of RDCs and scalar nuclear spin-spin couplings RDCs are grouped independent of the availability of resonance assignments -> more reliable fold recognition based on statistical analysis of RDC distributions Extended GFT NMR formalism / sampling protocol relaxes constraints thus far encountered for the design of GFT NMR experiments

62 Recent developments / projects Hi-fi NMR Automated Projection Spectroscopy Variations of FT for reconstruction Combination of GFT NMR with MEM, MDD Combination of GFT NMR with ultrafast NMR GFT solid state NMR

63 Frydman and Szyperski, 2003 From: NSF application submitted