SOLIDSTATE NMR STUDIES OF BIOMEMBRANES AND AMYLOID FIBRILS: The example of Synuclein

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SOLID-STATE NMR STUDIES OF BIOMEMBRANES AND
AMYLOID FIBRILS The example of ?-Synuclein
Jill Madine
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APPLICATIONS OF SOLID-STATE NMR
Biological membranes

• Protein-ligand interactions
• proton pump inhibitors
• digitalis receptor-ligand complex
• Protein-protein interactions
• phospholamban-SERCA association
• Protein-lipid interactions

Amyloid fibrils

• Intramolecular structure and
• intermolecular interactions
• a-synuclein
• amylin

Bucciantini, M. et al. Nature 416, 507-511
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SOLID-STATE NMR TECHNIQUES
Isotope labelling (15N, 13C, 19F) by chemical
synthesis or bacterial expression
Protein purification and preparation of membranes
Uniformly oriented
Random dispersion
magic angle spinning
interatomic distances
angles
membrane dynamics
protein and ligand structure
orientation of peptides in lipid bilayers
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?-SYNUCLEIN

• Lewy bodies and Lewy
• neurites are protein
• aggregates associated with neurodegenerative
  diseases
• Main protein component is ?-synuclein
• 140 amino acid presynaptic CNS protein with
  unknown function, suggestions include vesicular
  transport roles
• In solution it is unfolded, but aggregates into
  amyloid fibrils

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AIMS

• Gain understanding of the aggregation and
  membrane-binding properties of ?-synuclein using
  a domain-specific approach
• Understanding fibril formation/structure will
  enable potential inhibitors of aggregation to be
  studied
• Study relationship between membrane-binding and
  aggregation ? possible inhibitory role

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STRUCTURE
Bertoncini et al., PNAS, 2005
Solution
(10-48) N-terminal/membrane-binding
(120-140) C-terminal/Acidic
(71-82) Hydrophobic/Aggregation
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PEPTIDES
KTKEGV
1
2
3
4
5
6
7

• N-terminus similar to amphipathic a-helical
  domains of apolipoproteins
• 3 charge at neutral pH (8K, 5E)
• Structural studies indicate residues 1-102 form
  two helical regions with a short break at
  residues 43/44 - able to lie across membrane
  surfaces
• C-terminus is highly acidic (8- charge at neutral
  pH, 6E and 2D)
• Structural studies show it remains unfolded

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OVERVIEW OF WORK

• Membrane-protein interactions
• ?-syn(10-48), ?-syn(71-82) and ?-syn(120-140)
• Fluorescence
• Circular Dichroism
• 2H NMR
• 31P NMR
• Secondary structure determination of fibrils
• ?-syn(71-82)
• Electron Microscopy
• Congo Red
• 13C NMR

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MEMBRANE BINDING

• ?-Synuclein exists in equilibrium between soluble
  and membrane bound state
• Increase of 3-80 helicity upon membrane binding
• Membrane binding under tight control in vivo
• Membrane binding may be important in affecting
  fibrillisation rates (reduce or enhance)

DOPG negative
DMPC - neutral
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FLUORESCENCE BINDING ASSAY

• Addition of lipids to peptide solution quenches
  intrinsic tyrosine fluorescence emission at 305nm
  (following excitation at 280 nm)

• Increased quenching with mixed charged membranes

Preferential binding to neutral membranes
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CIRCULAR DICHROISM
TFE titration
DMPC/DOPG titration
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2H NMR

• Non-perturbing technique for characterising
  interactions between peptides/small molecules and
  phospholipid membranes
• 2H labels in polar head group of DMPC display
  pattern reflecting the quadrupole interaction of
  the deuterons within the magnetic field
• This splitting will change if the peptide
  interacts with the membrane surface and alters
  the lipid headgroup conformation

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2H NMR ?-SYN(10-48)
DMPC/DOPG
DMPC
?
? and ?
?
PeptideLipid
0
150
120

• Quadrupole splittings increase from 1.1kHz to
  2.2kHz (?) and decrease from 9.8kHz to 8kHz (?)
  upon peptide addition

• No significant changes in quadrupole splitting of
  5.7kHz (? and ?)

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2H NMR ?-SYN(71-82)
DMPC/DOPG
DMPC
PeptideLipid
0
150
120
Slight change in splittings indicate weak binding
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2H NMR ?-SYN(120-140)
DMPC/DOPG
DMPC
PeptideLipid
0
150
120

• Quadrupole splittings increase from 1.1kHz to
  2.3kHz(?) and decrease from 9.8kHz to 7.7kHz (?)
  upon addition of peptide

• One quadrupole splitting decreases from 5.7kHz to
  4.3kHz upon addition of peptide (? or ?)

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31P NMR

• 31P NMR used to monitor changes in phase
  behaviour of multilamellar lipid vesicles upon
  binding of ?-syn(10-48) and ?-syn(120-140)
• Phosphorus is present in both DMPC and DOPG
• Highly sensitive NMR nucleus making it an ideal
  non-perturbing probe of peptide-lipid
  interactions

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31P NMR
I homogeneous vesicles
II heterogeneous vesicles

• Peptide addition does not alter the overall
  bilayer structure

• Peptide addition causes change indicating phase
  separation

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CONCLUSIONS

• Membrane association of ?-synuclein is tightly
  regulated, with different regions of the protein
  involved in initiating interactions
• Induction of binding to negatively charged
  vesicles requires electrostatic interactions
  between N-terminal region, with acidic C-terminal
  residues favouring binding to neutral vesicles
• Other proteins acting in a similar way include
  apocytochrome c, cardiotoxin, myosin and
  antibacterial peptide PGLa

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?-SYN (71-82)

• Mutations within this region show decreased
  assembly
• ?-synuclein homologous except this region
• Resistant to proteolytic degradation
• Synthetic peptides self-polymerise into filaments
  (Giasson et al., Jbc, 2001)
• Binds to full length ?-synuclein to promote
  fibril formation

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AGGREGATION CONDITIONS

• 90?M ?-syn(71-82) incubated at 37C, in phosphate
  buffer for up to 6 weeks, with agitation

Scale bar 100nm
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13C NMR Line widths

• VTGVTAVAQKTV
• VTGVTAVAQKTV
• VTGVTAVAQKTV
• VTGVTAVAQKTV

? Line width indicates increase in order
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13C NMR Line widths
Peptide 1
Peptide 4
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CONCLUSIONS

• Greater ordering in the C-terminal residues upon
  fibril formation
• May represent core residues for fibrillisation

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FUTURE WORK

• Relationship between membrane-binding and
  aggregation
• Further study fibril structure to identify key
  residues involved in fibril formation (possible
  inhibition sites)
• Aggregation potential of a shorter hexamer
  peptide
• Potential inhibitors of ?-synuclein aggregation

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ACKNOWLEDGEMENTS

• David Middleton
• Andrew Doig
• Ashraf Kittmito
• People in labs G20 and G43
• Alzheimers Research Trust for scholarship and
  equipment grant