Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa

1. Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa Nobel 2002 Kurt Wüthrich

2. Proteiinien NMR-spektroskopia • Primaarirakenteen määrittäminen • Sekundaari ja tertiäärirakenteen tai konformaation määrittäminen • Kinetiikan ja molekulaarisen liikkeen tutkimus • Molekulaaristen vuorovaikutusten tutkimus

3. ? Rakenteen määrittämisenidea

4. Proteiinin NMR-spektri1957 Nobel E. Purcell 1952 Felix Bloch bovine pancreatic ribonuclease Saunders, Wishnia and Kirkwood

5. NMR-spektroskopian perusajatus Jokainen atomi, jolla on magneettinen ydin, antaa yksittäisen signaalin, joka sisältää informaatiota paikallisesta kemiallisesta ympäristöstä, rakenteesta ja dynamiikasta.

6. Magneettinen ydin Jokainen atomi, jolla on magneettinen ydin .... Stabiileja isotooppeja 1H, ~100% 13C, ~1.1% 15N, ~0.4% 31P, ~100% 2H, ~ 0% Pieni magneettinen momentti ~ sauvamagneetti

7. Miten valmistan NMR-näytteen? 1H, 31P-leimaus sellaisenaan • 13C, 15N-leimaus • Proteiinit: tuotetaan rikastetuista lähtöaineista: • 13C-glukoosi, 15N-ammonium suolat • DNA: PCR (lyhyet ketjut) • RNA: In vitro synteesi • 2H-leimausproteiini tuotetaan 2H-rikastetussa • mediumissa

8. Proteiinin NMR-spektri ... yksittäinen signaali ...

9. Paikallinen kemiallinen (~magneettinen) ympäristö • NMR-spektrometrin kenttä • paikallinen kenttä • -lähellä olevat ytimet • -ympäröivät elektronipilvet

10. Spektroskopian perusteista Energia kaavio – Purcellin kuva B/E

11. Resonanssispektroskopia B aika Fourier muunnos Vektorimalli – Blochin kuva taajuus

12. f1 t1 t2 f2 Kaksiulotteinen korrelaatiospektroskopia Signaalin modulaatio ajan t1 kuluessa S(t1, t2) S(f1, f2)  FT  Aikaulottuvuus  Fourier muunnos  taajuusulottuvuus (spektri)

13. NMR-spektroskopia kahdessa ulottuvuudessa 1991 Richard Ernst Jean Jeener

14. NMR of Biological MacromoleculesMultidimensional Multinuclear Spectroscopy Structural Biology

15. How to Interpret Spectra? ? • Structural implications • Atom type (and near neighbours) • Spatially near neighbours • Chemically bonded neighbours • Dynamic consequences • Fluctuating magnetic environment • Spectral parameters • Resonance frequency • Modulation of frequency • Correlation via dipolar field • Correlation indirectly via electrons (scalar coupling) • Relaxation

16. Magnetic EnvironmentDispersion of resonances • External magnetic field of the NMR-spectrometer • Local fields due to • -adjacent nuclei • -surrounding electron clouds • Chemical shift • = g(1 - s)B s is shielding (tensor)

17. Assignment of Resonances Proteins display large dispersion because they contain distinct magnetic microenvironments.

18. Assignment of ResonancesIdentification of Residues by Characteristic Chemical Shifts Aliphatic carbon shifts are particularly characteristic for the residues.

19. Assignment of Backbone ResonancesPrinciple – Sequential Walk HNCA H R H R H R | | | | | | -N–Ca– C –N– Ca– C –N– Ca– C- | || | || | || H O H O H O Ca S( Hi, Ni, Cai, Cai-1 ) N H HN(CO)CA H R H R H R | | | | | | -N–Ca– C –N– Ca– C –N– Ca– C- | || | || | || H O H O H O Ca S( Hi, Ni, Cai-1 ) N H

20. HNCA HN(CO)CA

21. Assignment of ResonancesSequential Walk via HNCO and HN(CA)CO

22. The redundancy in many alternatives for sequential assignment is important for automated assignment.

23. Spectra Contain Implict Structural DataNOEs  Short Distances NOEs Nuclear Overhauser Enhancement i.e. dipole-dipole relaxation.

24. Short Range Distances (NOEs) ri = rref(Sref/Si)1/6

25. Spectra Contain Implict Structural DataScalar Couplings  Dihedrals Karplus curve

26. How to Convert Spectral Parameters to Explicit Structural Data? • Short (<5-7Å) distances • via nuclear Overhauser spectroscopy (NOE) • Torsion angles • via scalar couplings (J-couplings) • Angles • via residual dipolar couplings (RDC) • Hydrogen bonds • via correlation spectroscopy • Secondary structures • via chemical shifts (resonance frequences)

27. T t Computation of Structure Conversion of structural data to restraints expressed as pseudo potentials Restrained molecular dynamics (MD) (Cartesian or torsion angle)

28. Result – Family of Structures All structures that satisfy restraints (within experimental error) are possible.

29. Evaluation of Structure • Accuracy • Restraint violations • Inconsitancies • Ramachandran violations • Precision • Spread of the family • Number of restraints • per residue

30. Direct Inspection of Spectra Observing binding Mapping binding epitopes Detecting conformational changes

31. About Field Fluctuations ”Reasons” ”Spectral Manifestations” • Bond vibrations • from pico to nano seconds • Conformational changes • from micro to milli seconds • Chemical exchange • from micro seconds to days • Relaxation measurements • -> rate constants, order • parameters, correlation times • Relaxation measurements • -> dispersion of parameters • Line width analysis • -> rate constants Motional model

32. Conformational Exchange

33. Conformational Exchange

34. Conformational Exchange

35. Relaxation Dispersion Transverse relaxation rates vs. effective field and temperature Frans A.A. Mulder et al.Nature Structural Biology 8, 932 - 935 (2001)

36. Hydrogen Exchange Monitoring signal intensity after dissolving to D2O Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,

37. Hydrogen Exchange Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,

38. Reaction Dynamics Elan Zohar Eisenmesser,1 Daryl A. Bosco,1 Mikael Akke,2 Dorothee Kern1* Science - Feb 2002,

39. Reaction DynamicsHot Spots

40. Reaction DynamicsProlyl Cis-Trans Isomerase

41. Dilute Liquid CrystalAnisotropic Medium

42. Distribution of Molecular Orientations

43. Vfree Net Alignment Vrestricted D = Dmax(3cos2q-1)/2

44. N A D C B Residual Dipolar Couplings

45. Alignments of Conformations

46. Monitoring Birth of Structure