characterizing a -synuclein membrane bound structure
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Characterizing a -Synuclein Membrane Bound Structure. Bert Lai (Gray Group) Department of Chemistry and Chemical Engineering California Institute of Technology Thesis Defense 04/29/08. Introduction: α-Synuclein. Role of α -syn in Parkinson’s disease pathogenesis unclear - PowerPoint PPT PresentationTRANSCRIPT
Characterizing -Synuclein Membrane Bound Structure
Bert Lai (Gray Group)Department of Chemistry and Chemical EngineeringCalifornia Institute of Technology
Thesis Defense04/29/08
Introduction: α-Synuclein • Role of α-syn in Parkinson’s disease pathogenesis unclear
• Aggregates form amyloid fibrils
• major component of Lewy Bodies deposits
• found in substantia nigra of Parkinson’s disease victims
• Three single point mutations
(A30P, A53T, and E46K)
• Early onset familial Parkinson’s
disease
Substantia nigra
Parkinson’s Disease
Normal
α-Synuclein Sequence
Three interesting regions:
1) Non β-Amyloid Component (NAC) region (Residues 61-95)
• Commonly found in Alzheimer’s disease aggregates
2) Acidic C-terminal region (Residues 96-140)
3) 7 imperfect repeats of 11 amino acids
• Second and seventh residues of each repeat are mostly KTKEGV
• Typically found in apolipoproteinsUlmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.
• Synaptic vesicle-associated protein
• Oligomers of α-syn involved in membrane permeabilization
• Structure:
• In solution: unfolded with structural preference
• In SDS micelles/acidic small unilamellar vesicles (SUVs):
highly helical
Introduction: α-Synuclein and Membrane
SDS micelles SUVs
1 nm20-50 nm
Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.
NMR Model of α-Synuclein in Micelles
2nd Helix (Lys45 – Thr92)
1st Helix (Val3 – Val37)
Linker (Lys38 – Thr44)
Hydrophilic Tail (Gly93 – Ala140)
• Formation of α-helices at
• residue 3 to 37 (1st helix)
• residue 45 to 92 (2nd helix)
• Formation of linker between 2 helices
• Unstructured hydrophilic tail
Phospholipid Vesicles
• EPR: Helical structure in the 7 N-terminal 11-amino acid repeats
Jao, C. C.; Der-Sarkissian, A.; Chen, J.; Langen, R. Proc Natl. Acad. Sci. 2004, 101 (22), 8331-8336.
Lipid exposed
Solvent exposed
The CD spectra show that acidic lipid is needed for α-syn to form helical structures.
Phospholipids
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC)
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphate (POPA)
1:1 POPC:POPA SUVs were made
For the N-terminus
Trp-Tyr(NO2) pairs
4
94
ro = 26 Å10 < r < 40 Å
W4-Y94
Helix #1
Helix #2
4
94
Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124 (19), 5481-5485.
FET Kinetics & Polypeptide Conformations
Steady State Fluorescence StudiesIn
tens
ity
W4 is in a more hydrophobic environment when associated with SDS or SUV
Wavelength (nm)
Amount of quenching by Y(NO2): SDS > SUV > Buffer
4
The two helices are closer together in SDS micelles. 94
4
NaPi
SDS
SUV
Distance (Å)
P(
r)
80 %
90 %
40 %
Time-resolved Fluorescence Studies
Time (ns)I t
SDS
SUV
Distance (Å)
P(
r)
Summary • Extended helix is elucidated when α-syn is in association with SUVs
• Similar to the EPR model
• α-syn forms compact structures when SDS micelles are introduced• Similar to the NMR model 94
4
Some extended structure remains
Calcium Binding Behavior of -Synuclein’s C-Terminal Tail
C-terminal Tail
Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Mol. Biol. 2005, 280, 9595-9603.
• locates in residues 96-140
• is unstructured in the presence of membrane mimic
• is highly negatively charged (10 Glus & 5 Asps)
• modulates aggregation
• binds to
• metal ions, e.g. calcium, copper
• microtubule-associated proteins 1B
• polyamines
Aging → Oxidative Stress → Calcium Dysregulation
Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.
de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.
Theory 1: C-terminal tail becomes β-sheet
Theory 2: C-terminal tail rigidified by binding calcium ion
Study C-terminal tail behavior in the presence of POPC:POPA with various Ca2+ concentration
Stage 1: Trp-only mutants
Stage 2: Donor-acceptor pair
DNP (N-Dinitrophenyl Phosphatidylethanolamine)
Br (6,7) POPC (1-Palmitoyl-2-Stearoyl(6,7-dibromo)-sn-Glycero-3-Phosphocholine)
McIntosh, T. J.; Holloway, P. W. Biochemistry 1987, 26, 1783-1788.
Quencher located on the end group
94 101
125 136
Headgroup/hydrocarbon boundary and quencher:3.5 Å
Trp Only Steady-state Fluorescence
NATA W125
W101 W136
Calcium Titration Curves (in SUVs)
[Ca2+] [Ca2+]
[Ca2+][Ca2+]
CD shows calcium ion does not affect the structure of α-synuclein.
CD
Time-resolved Fluorescent Lifetimes
W101 seems to be more associated with the membrane, but not deeply inserted.
W101 → DNP Fluorescence Transfer Rate
DNP + Ca2+
DNPA shift to shorter lifetimes is observed when calcium ions were added.
P(r
)
Log(k)
Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.
de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.
Theory 1: C-terminal tail becomes β-sheet
Theory 2: C-terminal tail rigidified by binding calcium ion
80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala
Acidic Residues
Mutation Sites
Ca Binding
Effective calcium binding has been shown for sequences DXXXD.
74
94101
125113
136
Seven mutants were expressed:
1) W94/Y113 6) W101/Y125
2) W94/Y125 7) W101/Y136
3) W94/Y136
4) W125/Y136
5) W101/Y74
Graphic made in PyMol
80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala
Acidic Residues
Mutation Sites
Ca Binding
Effective calcium binding has been shown for sequences DXXXD.
Binding sites for Calcium in solution is found between residue 101-113 & residue 125-136