Deakin Research Online This is the published version: Debeljuh, Natalie, Barrow, Colin J. and Byrne. Nolene 2011, The impact of ionic liquids on amyloid fibrilization of AB16-22: tuning the rate of fibrilization using a reverse Hofmeister strategy, Physical chemistry chemical physics, vol. 13, no. 37, pp. 16534-16536. Available from Deakin Research Online: http://hdl.handle.net/10536/DRO/DU:30039443 Reproduced with the kind permission of the copyright owner. Copyright: 2011, Royal Society of Chemistry.

16534 Phys. Chem. Chem. Phys., 2011, 13, 16534–16536 This journal is c the Owner Societies 2011

Cite this: Phys. Chem. Chem. Phys., 2011, 13, 16534–16536

The impact of ionic liquids on amyloid fibrilization of Ab16-22: tuningthe rate of fibrilization using a reverse Hofmeister strategyw

Natalie Debeljuh,aColin J. Barrow

band Nolene Byrne*

a

Received 11th July 2011, Accepted 3rd August 2011

DOI: 10.1039/c1cp22256b

We have shown that the amyloid fibrilization of Ab16-22 followsa reverse hofmeister trend in pILs. Fast fibrilization rates of

seconds can be achieved.

The self assembly and subsequent amyloid fibril formation of

proteins has implications in many human diseases, including

Alzheimer’s disease.1,2 The pathogenesis of Alzheimer’s disease

involves the self assembly of Ab monomers into dimers,

multimers and oligomers.1,3 The oligomers self assemble into

higher order aggregates termed protofilaments which continue

to grow into fibrils.4 The processes of Ab peptide assembly

from monomers to oligomers and into fibrils, and their

associated neurotoxicity is still an active area of research.5–8

Currently the neurotoxic species in the amyloid fibril assembling

process is thought to be some form of soluble oligomer, most

likely those that form during the early stages of Abaggregation.7,9,10 Recent trends have been directed at finding

strategies to control the Ab assembly process to allow

characterization of the early state oligomers.9,11,12 In addition

to developing methods to trap the early state oligomers, the

development of drugs specifically targeted at early stage Abaggregation are currently under investigation.12

Solvents can play a key role in controlling the fibrilization of

Ab,13,14 as such, various solvents and additives have been used

to study to Ab amyloid fibrilization. Ionic liquids are designer

solvents comprised entirely of ions. The advantages of ionic

liquids as solvents include low volatility, recyclability, excellent

solvating properties, variable polarity and a tremendous selection

of cation and anion combinations.15,16 Since the formation of

amyloid fibrils proceeds via the formation of intramolecular

hydrogen bonds we sort to use protic ionic liquids to manipulate

the hydrogen bond nature of the solvent to better control

amyloid fibril formation. The use of Ionic liquids as solvents

for biological applications is currently receiving considerable

attention, and ionic liquids have been used successfully as

refolding additives for protein renaturing studies,17,18

increased protein shelf life,19 enhanced thermal stability20,21

and as additives which increase enzymatic reaction rates.22

Recently pILs where shown to both promote and

inhibit amyloid fibrilization of hen egg white lyszyme.23,24

Hwang et al. also reported the ability of 1-Butyl-3-methylimidazolium

bis(trifluoromethylsulfonyl)imide to accelerate the fibrilization

of a-synuclein.25 Therefore, the characterization of ionic liquid

solvents and the impact they have on amyloid fibrilization is

important, especially since ionic liquids potentially have an

important role to play in furthering our knowledge of the

amyloid fibrilization process and in drug design. In this

context, our aim was to study the influence pILs have on the

amyloid fibrilization of the Ab16-22 peptide. This fragment

has been previously identified as a core fragment for amyloid

fibril formation in full length Ab,26,27 making this fragment an

appropriate peptide sequence to further our understanding of

the role ionic liquids can play in amyloid fibrilization.

We have used a combination of ThT fluorescence intensity,

circular dichroism, and transmission electron microcopy to

analyze and characterize the amyloid fibrilization of the Ab16-22peptide using protic ionic liquid solvents.

The kinetics of amyloid fibrilization can be measured using

the ThT binding assay. ThT fluorescence intensity is sensitive

to beta sheet structure, with an increase in the fluorescence

intensity being measured when aggregation have proceeded to

the organized amyloid stage. Fig. 1 shows the ThT intensity

kinetics of the Ab16-22, in a series of different pILs with the

common cation triethylammonium, (Tea). pILs investigated in

Fig. 1 ThT intensity as a function of time for Ab16-22 in 90%

TeaHSO4 (red curve), 90% TeaH2PO4 (blue line), 90% TeaTFac

(green line), 90% TeaLa (purple line), 90% TeaTf (light blue line),

90% TeaMs (orange line) and phosphate buffer (black line).

a Institute for Technology Research and Innovation,Deakin University, Geelong, Vic 3217, Australia.E-mail: nolene.byrne@deakin.edu.au

b School of Life and Environmental Sciences, Deakin University,Geelong, Vic 3217, Australia

w Electronic supplementary information (ESI) available: Materialsand methods; ESI Fig. 1; movie clip capturing fast fibrilization. SeeDOI: 10.1039/c1cp22256b

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this study include, triethylammonium phosphate (TeaH2PO4),

triethylammonium hydrogen sulfate (TeaHSO4), triethylammonium

trifluoroacetate, (TeaTfac), triethylammonium lactate,

(TeaLa), triethylammonium triflate, (TeaTf), and triethylammonium

mesylate, (TeaMs). Since both TeaH2PO4 and TeaHSO4 have

melting points above room temperature, we measured the ThT

intensity of the Ab16-22 peptide from 97%TeaH2PO4:3%water

(v/v) through to 5%TeaH2PO4:95%water (v/v) and found that

90% TeaH2PO4 showed fast kinetics (ESI Fig. 1w). Since we

are interested in high pIL content solvents we therefore

decided to compare the kinetics of fibrilization in the above

mentioned pILs at a fixed concentration of 90%pIL:10%water

(v/v). The ThT intensity of the Ab16-22 peptide in different

pILs is shown in Fig. 1. Both TeaH2PO4 and TeaHSO4,

showed remarkably fast fibrilization with the maximum ThT

signal observed after 1 min. The kinetics of both TeaTfac

and TeaLa show a lag phase with fibrilization proceeding to

a measured maximum after several hours for TeaTfac and

2 days for TeaLa. TeaTf showed a longer lag phase with a

maximum ThT signal observed after 2 weeks. In the cases of

TeaTfac, TeaLa and TeaTf, the maximum ThT signal is less

than 1, this may indicate either the presence of aggregated

states or a different amyloid structure. Interestingly, no fibrils

formed in the 90%TeaMs solution even after 4 months,

and more importantly the solution remained clear indicating

the Ab16-22 was still in solution. No change in secondary

structure, as measured using CD, was observed over this time

period, Included in Fig. 1 is the ThT response for Ab16-22 in

phosphate buffer, which reached a maximum on day 9.

A reverse Hofmeister trend is observed when the pILs are

listed in order of fibrilization rate. That is, TeaHSO4, Tea

H2PO4, TeaTFac, TeaLa, TeaTf, and TeaMs. Traditionally,

both phosphate and sulphate are considered to be kosmotropic

anions. These anions are considered to be water structuring

and generally enhance protein stability.28 The reverse Hofmeister

trend observed here in the 90% pIL solutions suggests that

competitive hydrogen bonding between the anion of the pIL

and water is driving the self assembly of the Ab peptide into

amyloid fibrils. In this context both TeaH2PO4 and TeaHSO4

are ‘‘salting out’’ the Ab peptide and resulting in amyloid

fibrils. It also suggests that in high pIL concentration solutions

such as the ones studied here (90%pIL;10%water (v/v)), does

not completely dissociate into individual ions, and probably

ion pairs or higher order aggregates exist or the presence of

microsegregated IL phase,29,30 This would explain the deviation

from the Hofmeister series, which is often used to explain the

salting in and salting out effects of salts with respect to protein

stability and solubility.

The fast kinetics of fibrilization in 90%TeaH2PO4 confirmed

by the ThT binding experiment supports our initial observation

of white participates forming in the 90%TeaH2PO4 seconds

after the monomeric peptide is added. (ESI shows movie clip

of fibrilizationw). We then decided to examine the fibrils using

TEM, long mature fibrils were present in the solution after one

minute as shown in Fig. 2a. Fig. 2b shows an overview of the

amyloid fibrils that can be achieved after one minute in

90%TeaH2PO4. A dense highly branched network is observed.

This extremely fast fibrilization rate holds considerable

promise in the application of amyloid fibrils for biomaterials.

Fig. 3a shows the micrograph of the Ab16-22 peptide, after

1 min, in 90%TeaHSO4. This pIL also registered a maximum

ThT intensity after one minute. However, as can be seen from

the micrograph, in this case fibrils are not present, rather

spherical objects can be seen in the micrograph. The spherical

objects, are termed annular oligomers.31 The formation and

stabilization of annular oligomers is an important finding as it

has been suggested that annular oligomers may be wholly or

partly responsible for the cytoxicity associated with the formation

of amyloid fibrils.11,30 The annular oligomers are stabilized by

the TeaHSO4 pIL for 2days after which small fibrils can be

seen, however some spherical objects are still present (Fig. 2b).

The presence of the annular oligomers and amyloid fibrils in

the same solution supports the complexity of amyloid

fibrilization process and why pinning down the toxic confor-

mation is difficult.

Fig. 4 shows the CD spectrum of Ab16-22 for 90%Tea-

HSO4 and 90%TeaMs. Given the difference in fibrilization

kinetics observed in the pIL solutions we measured the CD

spectrum of Abeta in 90%TeaHSO4 and 90%TeaMs.

Fibrilization in 90%TeaHSO4 occurs on the time scale of

minutes whereas no fibrilization is observed in 90%TeaMs.

Interestingly the conformation of the peptide in both these

solutions is very similar. The similarity of the CD spectrum,

which represents an extended 310helix,32 suggests that the

Fig. 2 TEM images shows amyloid fibrils of the Ab–22 in

90%TeaH2PO4 at 1 min and (b) overview of (a) showing a dense

amyloid network after 1 min. Scale bar 200 nm.

Fig. 3 TEM images showing amyloid fibrils of Ab16-22 in

90%TeaHSO4 at (a) 1 min and (b) 2 days. Scale bar 200 nm.

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16536 Phys. Chem. Chem. Phys., 2011, 13, 16534–16536 This journal is c the Owner Societies 2011

initial conformations, for the Ab16-22 peptide in both these

solutions is similar. This further suggests that the anion-water

interactions of the pIL is the driving force for the differences

observed in the fibrilization kinetics.

We have shown that the kinetics of fibrilization for Ab16-22can be tuned by following a reverse Hofmeister strategy in pIL

solutions using the triethylammonium cation. Fibrilization

can be both accelerated or completely suppressed depending

on pIL choice. This has potential impact in understanding the

underlying process of amyloid fibrilization. Additional, the

ability to create fibrils on the time scale of seconds is important

from a biomaterials aspect. The use of amyloid fibrils to create

nanostructures is a promising avenue of research33 and the

ability to accelerate fibrilization is beneficial in this context.

The kinetics of fibrillization shown here follows a reverse

Hofmeister trend. When pILs contain kosmotropic anions

such as phosphate the rate of amyloid fibrilization is increased,

in a ‘‘salting out’’ scenario, while pILs containing mesylate

anions suppress amyloid fibril formation, in a ‘‘salting in’’

scenario. One possible explanation for this observation is that

strong anion–water interactions are driving the self assembly

process. The structure of water involves ice-like structure

pools and disordered zones. The addition of highly ordered

anions such as phosphate or sulfate may cause the disordered

zones to become more ordered as a result of H-bonding

between the anion and water, resulting in an increase in the

volume occupied by the ions. This competitive H-bonding

between the water and the anion may, in the protic ionic liquid

case, be driving the self assembly of the Ab16-22 into amyloid

fibrils.

The stabilization of annular oligomers is an important

finding and future work is aimed at investigating the

neurotoxicity of the ionic liquid stabilized states and the ability

for ionic liquids to reverse the aggregation process of amyloid

fibrils from the Ab16-22 peptide.

Conclusions

We reported the fibrilization kinetics and superstructure

organization of the Ab16-22 peptide in a series of protic ionic

liquids (pIL). The kinetics of Ab16-22 fibrilization follows a

reverse Hofmeister trend. We find that pILs containing

kosmotropic anions like phosphate or sulphate promote

fibrilization with mature fibrils forming within seconds whilst

pILs containing the mesylate anion completely suppresses

amyloid fibrilization. The self assembly and superstructure

organization of the amyloid fibrilization process was also

found to be influenced by pIL composition, resulting in the

stabilization of annular oligomers, a key intermediate state.

The authors would like to thank the Centre for Biotechnology,

Chemistry and Systems Biology for financial assistance. NB

acknowledges the Australian Research Council for an APD.

Notes and references

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Fig. 4 CD spectrum of Ab16-22 in 90%TeaMs (black) and

90%TeaHSO4 (red).

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