Sheng Zhang, Stan Yoo, Adam G. Kreutzer, James S. Nowick

Department of Chemistry, University of California, Irvine

The Expression and Characterization of

Disulfide-bond Stabilized Amyloid-β peptides

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qAmyloid-β (Aβ)

v Central to the pathogenesis of Alzheimer’s disease

qAβ oligomers

v Toxic

v Aggregation-prone

Kreutzer, A. G. et al. J. Am. Chem. Soc. 2017, 139, 966–975.

Knowles, T. P. J. et al. Nat. Rev. Mol. Cell Biol. 2014, 15, 384–396.

Vivekanandan, S. et al. Biochem. Biophys. Res. Commun. 2011, 411, 312.

http://pdb101.rcsb.org/motm/189

Research gap: Aβ oligomers are challenging to study

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q β-hairpin → Aβ oligomers

q Conformational change → Aβ fibrils

Hoyer, W. et al. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5099–5104.

Sandberg, A. et al. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15595–15600.

Inspiration: β-hairpin is required for Aβ oligomerization

PDB ID: 2OTK

White: affibody

Green: Aβ β-hairpin

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Aβ (M1–42/A21C–A30C) (mutant 1)

Research goal: study effects of β-hairpin alignment of Aβ

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(mutant 2)

(mutant 3)

(mutant 4)

(mutant 5)

Mutant Aβ plasmids were generated through molecular cloning

Nde1/Sac1

Aβ WTNde1

Sac1

Nde1

Sac1

Aβ mutant Nde1/Sac1

Nde1 Sac1

SAP

Aβ mutant

Nde1 Sac1

T4 ligase

Nde1

Sac1

Aβ mutant

Amp Amp

Amp

WT plasmid

mutant plasmid

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Disulfide-bond stabilized Aβ were expressed and purified

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Representative HPLC:

Aβ (M1–42/A21C–I32C)

(mutant 2)

Representative mass spec:

Aβ (M1–42/A21C–I32C)

(mutant 2)

Disulfide-bond stabilized mutants like to form oligomers

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kDa

SDS-PAGE results of Aβ (M1–42) wild-type and

disulfide-stabilized peptides (at 31.25 µM)

Disulfide-bond stabilized mutants do not form fibrils

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time (h)

AVG_AβM42_10µM AVG_AβM42_Mut1 AVG_AβM42_Mut2

AVG_AβM42_Mut3 AVG_AβM42_Mut4 AVG_AβM42_Mut20Mut 3

WT Mut 1 Mut 2

Mut 4 Mut 5

ThT assay results of Aβ (M1–42) wild-type and

disulfide-stabilized peptides (at 10 µM)

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Reduction of the disulfide-bond induced the formation of the fibrils

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ThT assay results of Aβ (M1–42/A21C–I32C) in the absence or

presence of TCEP reducing agent (at 10 µM)

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0 1 3 4 5 7 8 9 11 12 13 15 16 17 19 20 21 23 24

10 µM Aβ(M1–42/A21C–I32C) (mut 2) + 5 mM TCEP10 µM Aβ(M1–42/A21C–I32C) (mut 2) + water

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time (h)

Disulfide-bond stabilized mutants – circular dichroism

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Disulfide-bond stabilized mutants – ATR-FTIR

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β-turn

β-sheet

𝛼-helix

Acknowledgments

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o Professor James S. Nowick and the Nowick lab members

o National Institutes of Health (NIH)

o UCI Mass Spectrometry facility (Ben Katz and Felix Grun)

o UCI Laser Spectroscopy facility (Dmitry Fishman and Christian Baca)

o The Martin, Tsai, Spitale, Weiss, and Prescher labs at UCI