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Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR“ Presented by: Daniel Droste 17.10.2013 Seminar Magnetische Resonanz
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Page 1: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

Bayro, 2011, JACS„Intermolecular structure determination of

amyloid fibrils withmagic-angle spinning and

dynamic nuclear polarization NMR“

Presented by: Daniel Droste17.10.2013

Seminar Magnetische Resonanz

Page 2: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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

Page 3: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

Page 4: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

PI and SH3 domain of PI3

Römpp online [Elektronische Ressource] : der effizientere Zugriff auf das Wissen der Chemie, Stuttgart : Thieme, 2001-

Src(cellular und sarcoma)-homology 3 domain of the Phosphatidylinositide 3-kinase„Biological Assembly Image for 3I5SCrystal structure of PI3K SH3Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient“,http://www.rcsb.org/pdb/images/3i5s_bio_r_500.jpg, 12.10.2013

Page 5: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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

Page 6: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

Magic Angle Spinning

● Abbrev. MAS● improves signal quality● removes anisotropic

interactions by averaging

● magic angle θm=54,75°

to magnetic field

● (cosθm)²=1/2

http://www.magnet.fsu.edu/education/tutorials/tools/probes/images/probes-mas-angle5.jpg, 15.10.2013

Page 7: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

BASE RFDR

● without selection: correlation between aliphatic nuclei in distance too weak to be observed

● band selective radio frequency dipolar recoupling

Angewandte Chemie International EditionVolume 48, Issue 31, pages 5708-5710, 27 JUN 2009 DOI: 10.1002/anie.200901520http://onlinelibrary.wiley.com/doi/10.1002/anie.200901520/full#fig1

„Schematic representation of pulse phases and durations asa 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.

Page 8: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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

Page 9: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 084508 (2012).

Page 10: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

(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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

K=LysineG=GlycineT=ThreonineF=PhenylalanineY=TyrosineD=Aspartic acid

Page 11: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

Page 12: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

Section of a BASE RFDR spectrum of amyloid fibrils formed by 2-PI3-SH3. Gray labels indicate sequential 13Cα–13Cα cross-peaks 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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

M=MethionineA=AlanineR=ArganineD=Aspatic acidF=PhenylalanineE=Glutamatic acidS=SerineY=Tyrosine

Page 13: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

D=Aspartic acidN=Asparagine

Page 14: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

missing resonances

300K,750 MHz

100K,400MHz

carbonyl aromatic aliphatic

Page 15: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

(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, 13967-13974.DOI: 10.1021/ja203756xCopyright © 2011 American Chemical Society

Page 16: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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

Page 17: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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

Page 18: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

Outlook

● position of the turn of the ß-sheets: more verification

● more interactions between protein● additional constraints:

– DNP● higher dimensional● higher field

Page 19: Bayro, 2011, JACS „Intermolecular structure determination of … · 2013-10-18 · Bayro, 2011, JACS „Intermolecular structure determination of amyloid fibrils with magic-angle

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, 943–947 (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, 084508 (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, 064506 (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, 13967–13974 (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, 17077–17079 (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, 5708–5710 (2009).


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