Joshua T.B. Williams*1, Laura Luther*2, Andrew J. Hawk3, Joseph R. Sachleben4, Stephen C. Meredith1,3

*Contributed equally to this project; 1Pritzker School of Medicine, The University of Chicago, Chicago IL 60637; 2Department of Chemistry;3Departments of Biochemistry and Molecular Biology, The University of Chicago; 4Biomolecular NMR Facility, Shared Facilities, The University of Chicago

Chaotic Aggregation of !-Amyloid Congener Peptides

Introduction

Two broad hypotheses for aggregation of amyloid beta (A!)

peptides exist. One category suggests that an autonomouslyfolding core domain of the peptide, possibly A!21-30, initiates

aggregation into !-sheet-rich oligomers and fibrils. This structure

would allow flanking hydrophobic residues to associate with eachother and eventually form !-sheet in fibrils. The alternative

category posits that interactions between hydrophobic residuesflanking the flexible, hydrophilic domain of A!21-30 cause

polymorphic associations that propagate self-association into

oligomers and fibrils.

In order to compare these hypotheses, we examined severalinternal A! core fragments – A!21-30, A!16-34, and A!13-38 – as well

as A!21-30 or A!16-34 extended at their N-termini by a Cys residue,

or as cyclic peptides. The Cys-containing or cyclic peptides, we

hypothesized, might stabilize weak interactions between sidechains in A!21-30 through an entropic effect, rendering them more

manifest by NMR spectroscopy and other techniques.

Size Exclusion Chromatography

SEC with void and total volume markers. Black (1): BSA. Red(2): synthetic A!1-40. Dark blue (3a&b): dimeric and monomeric

Cys-A!16-34, respectively, in the presence of TCEP. Orange

(4a&b): dimeric and monomeric Cys-A!21-30, respectively, in the

presence of TCEP. Light blue (5): A!16-34. Green (6): A!21-30.

Yellow (7): Glycine. A!21-30 is entirely monomeric in wild type and Cys-modified

variants. Cys-A!16-34 forms disulfide-bonded dimers and a small

amount of soluble oligomer that elutes in the void volume.

A! Peptide Sequences

Serial SEC of a

fibrillizing solution of Cys-A!16-34 shows depletion

of soluble oligomers first,

then dimers, and finally

monomers.

Although compatible

with other schemes, this

suggests that monomers

or dimers are sufficient

for fibril formation.

Peptide solutions were freshly prepared at 100!M (black) to 500!M(pink) and examined between 180-280nm. A!21-30 is unstructured. A!16-34

and A!13-38 develop a minimum at ~228 nm, compatible with a !-turn

structure.

A!16-34 A!13-38A!21-30

Electron micrographs of fibrils of various synthetic peptides. (A) A!21-30

formed no fibrillar material. (B) A!16-34 in phosphate buffer, pH 7.40. (C) A!

13-38 in phosphate buffer, pH 7.40. (D) Cys-A!16-34, allowed to dimerize in

neat DMSO, before transfer to citrate buffer, pH 3.50. (E) A!16-34 fibrils,

formed by seeding with dimeric Cys-A!16-34. (F) Cyclo-Cys-A!16-34, initially

dissolved in DMSO, before transfer to citrate buffer, pH 3.50. Thioflavin T

analysis of fibrils demonstrates that dimerization increases the rate and

extent of fibril formation.

Circular Dichroism

Electron Microscopy

A B C

D E F

Nuclear Magnetic Resonance Spectroscopy

Figures: (I) 2D-1H TOCSY spectra of A!16-34 at pH 4-7. (II)

Selective signal (volume) loss in individual peaks in sequentialNOESY spectra of A!16-34; average of three spectra. (III) 2D-1H

TOCSY spectra of Cys-A!16-34 at pH 3-7. (IV) Selective signal

(volume) loss in individual peaks in sequential TOCSY spectra ofA!16-34; average of three spectra.

I

II

III

IV

Conclusions

We find no evidence that A!21-30 constitutes an autonomously folding

core domain or has the ability to promote oligomerization and fibrillizationof A! monomers. Conversely, extended A! peptides - A!16-34, A!13-38

-

possess a !-turn-like structure and self-aggregate into soluble oligomers

and fibrils. Furthermore, their N-terminal Cys variants favor interactionsbetween sidechain residues known to interact in A! fibrils. In Cys-A!16-34,

fibrils form pari passu with the formation of disulfide bonds.Finally, sequential NMR of A!16-34 and Cys-A!16-34 demonstrate non-

uniform signal decay, indicating a failure to adopt a single structure and

the formation of polymorphic aggregated species (i.e. oligomers and fibrils)

favored by the hydrophobic effect.

Acknowledgements

This work was made possible by R01-NS04252 and T32-HL007327.

We thank Yimei Chen for assistance with Electron Microscopy and

Elena Solomaha for assistance with Circular Dichroism.