Bayro, 2011, JACS Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

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1 Bayro, 2011, JACS Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR Presented by: Daniel Droste Seminar Magnetische Resonanz

2 Outline 1. Principal result 2. Phosphatidylinositol 3. PI and SH3 domain of PI3 4. NMR methods 5. Magic Angle Spinning 6. BASE RFDR 7. Advantages of BASE RFDR 8. Dynamic Nuclear Polarization (DNP) 9. Spectra 10. Summary 11. Applications 12. Outlook 13. Further Literature

3 Principal result strands of the Src(cellular und sarcoma)homology 3 domain of the Phosphatidylinositide 3-kinases are parallel and in register Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

4 PI and SH3 domain of PI3 Römpp online [Elektronische Ressource] : der effizientere Zugriff auf das Wissen der Chemie, Stuttgart : Thieme, Src(cellular und sarcoma)-homology 3 domain of the Phosphatidylinositide 3-kinase Biological Assembly Image for 3I5S Crystal structure of PI3K SH3 Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient,

5 NMR methods 1.BASE RFDR: detection of 13Cα 13Cα contacts 2.comparison of 13Cα 13Cα correlations 3.low-temperature DNP heteronuclear spectroscopy using mixed 15N/13C

6 Magic Angle Spinning Abbrev. MAS improves signal quality removes anisotropic interactions by averaging magic angle θm=54,75 to magnetic field (cosθm)²=1/

7 BASE RFDR without selection: correlation between aliphatic nuclei in distance too weak to be observed band selective radio frequency dipolar recoupling Angewandte Chemie International Edition Volume 48, Issue 31, pages , 27 JUN 2009 DOI: /anie Schematic representation of pulse phases and durations as a function of pulse pair index for [...] decoupling sequences: (a) TPPM Vinod Chandran, C., Madhu, P. K., Kurur, N. D. & Bräuniger, T. Swept-frequency two-pulse phase modulation (SWf-TPPM) sequences with linear sweep profile for heteronuclear decoupling in solid-state NMR. Magn. Reson. Chem. 46, 944 (2008), Fig. 1.

8 Advantages of BASE RFDR 1.stable when the experimental conditons are not perfect 2.low 13C power levels no depolarization processes no heteronuclear interference 3.recoupling by finite pulses 4.fewer unwanted 13Cα(i) 13C (i 1) polarization transfer 5.attenuation of dipolar truncation

9 Dynamic Nuclear Polarization (DNP) transfer of magnetization from electron on nuclei microwaves use of organic radicals interactions: Cross effect Solid effect Nuclear Overhauser Effect Thermal mixing effect Thurber, K. R. & Tycko, R. Theory for cross effect dynamic nuclear polarization under magic-angle spinning in solid state nuclear magnetic resonance: The importance of level crossings. J. Chem. Phys. 137, (2012).

10 K=Lysine G=Glycine T=Threonine F=Phenylalanine Y=Tyrosine D=Aspartic acid (a) Subsection of a BASE RFDR spectrum of microcrystalline 2-GB1 showing cross-peaks between Y45Cα and neighboring nuclei. (b) Internuclear distances in the crystal structure of GB1 (PDB ID 2QMT) corresponding to the cross-peaks observed between Y45Cα and other 13Cα sites, i.e., within its own strand (T44, D46, and D47), to a strand within the same molecule (T51 and F52), and to an adjacent strand in a neighboring molecule (K13* and G14*). Asterisks denote residues in an adjacent protein molecule in the crystal lattice. The spectrum in panel a was recorded with τmix = 24 ms and a total experimental time of 7.5 h. Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

11 Internuclear distances anticipated in parallel β-strands and resolvable 13Cα 13Cα correlations for a given residue in the middle of three different strands, h, i, k (left), and three identical in-register strands, i, i, i (right). Interstrand correlations in the parallel in-register case are degenerate with sequential correlations within the strand. Typical internuclear distances are indicated on the left. Dashed lines of different colors (except for black) indicate the potentially resolved cross-peaks in 13C 13C correlation spectra. Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

12 M=Methionine A=Alanine R=Arganine D=Aspatic acid F=Phenylalanine E=Glutamatic acid S=Serine Y=Tyrosine Section of a BASE RFDR spectrum of amyloid fibrils formed by 2-PI3-SH3. Gray labels indicate sequential 13Cα 13Cα crosspeaks while black labels denote cross-peaks between 13Cα nuclei separated by two residues, with an internuclear distance corresponding to 6.5 Å. Backbone backbone correlations between sites distant in space, but near in sequence, are readily observed for several regions of the polypeptide chain. This spectrum was recorded with τmix = 24 ms and a total experimental time of 5 days. Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

13 D=Aspartic acid N=Asparagine Sections of PDSD 13C 13C correlation spectra acquired with a mixing time of 20 ms optimized for one-bond correlations of (a) U-PI3-SH3 and (b) 2-PI3-SH3, and with a mixing time of 500 ms optimized for long-range correlations in (c) 2-PI3-SH3. The dotted boxes in panels a and b correspond to the same region as that shown in panel c, in which asterisks identify correlations between neighboring molecules in a parallel, in-register architecture. Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

14 missing resonances 300K, 750 MHz carbonyl aromatic aliphatic 100K, 400 MHz Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

15 (a) Summary of intermolecular constraints along the PI3-SH3 sequence obtained with the methods described in the text: Indirect CC ( > ), direct CC ( * ), mixed NC at room temperature ( ), and mixed NC at 100 K with DNP ( + ). Filled bars indicate residues in a β-strand conformation while empty bars mark dynamic regions that have not been assigned in the spectra. (b) Superposition of all intermolecular constraints on a hypothetical model of PI3-SH3 amyloid fibril architecture in which two β-sheet layers (light gray and dark gray, respectively) are formed by each half of the sequence. Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas; Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, DOI: /ja203756x Copyright 2011 American Chemical Society

16 Summary -verification of parallel, in-register ß-sheet structure in amyloid fibrils (emme-loid) -sample used: PI3-SH3, immunoglobulin protein G 1. BASE RFDR: 2-13C glycerol: 13C-13C contacts between adjacent strands and neighboring molecules for clearing structural degeneracy 2. comparison of short- and long-range 13C-13C correlations : distinguish between intra- and inter residue contact, mutually exclusive 13C-12C and 12C-13C 3. low temperature DNP: 15N-13C temperature; big advantage of S/N, quenching of dynamics

17 Applications amyloid fibrils may be a cause for Alzheimer's disease and type 2 diabetes results at low temperatures of otherwise missing resonances possibility for the determination of supramolecular interactions in general

18 Outlook position of the turn of the ß-sheets: more verification more interactions between protein additional constraints: DNP higher dimensional higher field

19 Further Literature 1. Vinod Chandran, C., Madhu, P. K., Kurur, N. D. & Bräuniger, T. Swept-frequency two-pulse phase modulation (SWf-TPPM) sequences with linear sweep profile for heteronuclear decoupling in solid-state NMR. Magn. Reson. Chem. 46, (2008). 2. Thurber, K. R. & Tycko, R. Theory for cross effect dynamic nuclear polarization under magic-angle spinning in solid state nuclear magnetic resonance: The importance of level crossings. J. Chem. Phys. 137, (2012). 3. Bennett, A. E. et al. Homonuclear radio frequency-driven recoupling in rotating solids. J. Chem. Phys. 108, 9463 (1998). 4. Tycko, R. Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR. J. Chem. Phys. 126, (2007). 5. Bayro, M. J. et al. Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR. J. Am. Chem. Soc. 133, (2011). 6. Debelouchina, G. T., Platt, G. W., Bayro, M. J., Radford, S. E. & Griffin, R. G. Intermolecular Alignment in β2-microglobulin Amyloid Fibrils. J. Am. Chem. Soc. 132, (2010). 7. Bayro, M. J., Maly, T., Birkett, N. R., Dobson, C. M. & Griffin, R. G. Long-Range Correlations between Aliphatic 13C Nuclei in Protein MAS NMR Spectroscopy. Angew. Chem. Int. Ed. 48, (2009).