Organic solvent mediated self-association of an amyloid forming peptide from β2-microglobulin: An atomic force microscopy study

Organic Solvent Mediated Self-Association of an Amyloid FormingPeptide From b2-Microglobulin: An Atomic Force Microscopy Study

Nitin Chaudhary, Shashi Singh, Ramakrishnan NagarajCentre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road,

Hyderabad 500 007, India

Received 18 February 2008; revised 9 August 2008; accepted 3 September 2008

Published online 17 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21087

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

Alarge number of peptides corresponding to seg-

ments of amyloid forming proteins show the ability

to form fibrillar structures.1–19 This property is also

observed in de novo designed peptides.20,21 The

peptides have the ability to form mature fibrils that

cause an increase in thioflavin T (ThT) fluorescence. The

conditions for the formation of fibrils by peptides are highly

variable with respect to pH, temperature, and incubation pe-

riod. A common structural feature in amyloid fibrils formed

by peptides and proteins is an extensive b-sheet network sta-

bilized by backbone hydrogen bonds.3,14,18,22,23

The protein b2-microglobulin (b2m), which constitutes

the light chain of the major histocompatibility antigen class

1, forms amyloid fibrils in patients with renal failure.24 The

protein also forms amyloid fibrils in vitro under a variety of

conditions.25–31 Several peptides spanning the beta strand

regions of b2m have been analyzed for their ability to form

fibrils.7,13,16 Two peptides DWSFYLLYYTEFT and DWSFYL-

LYYTEFTPTGKDEYA showed extensive fibril growth over a

wide pH range and ionic strength as deduced from ThT fluo-

rescence and electron microscopy.7 The shorter peptide

Organic Solvent Mediated Self-Association of an Amyloid FormingPeptide From b2-Microglobulin: An Atomic Force Microscopy Study

Correspondence to: Ramakrishnan Nagaraj; e-mail: [email protected]

ABSTRACT:

Human b2-microglobulin (b2m) forms amyloid fibrils in

hemodialysis related amyloidosis. Peptides spanning the

b strands of b2m have been shown to form amyloid fibrils

in isolation. We have studied the self-association of a 13-

residue peptide Ac-DWSFYLLYYTEFT-am (Pb2m)

spanning one of the b-strands of human b2-

microglobulin when dissolved in various organic solvents

such as methanol (MeOH), trifluoroethanol (TFE),

hexafluoroisopropanol (HFIP), and dimethylsulfoxide.

We have observed that Pb2m forms amyloid fibrils when

diluted from organic solvents into aqueous buffer at pH

7.0 as judged by increase in thioflavin T fluorescence.

Fibril formation was observed to depend on the solvents

in which peptide stock solutions were prepared. Circular

dichroism spectra indicated propensity for helical

conformation in MeOH, TFE, and HFIP. In buffer, b-

structure was observed irrespective of the solvent in which

the peptide stock solutions were prepared. Atomic force

microscopy images obtained by drying the peptide on

mica from organic solvents indicated the ability of Pb2m

to self-associate to form nonfibrillar structures.

Morphology of the structures was dependent on the

solvent in which the peptide was dissolved. Peptides that

have the ability to self-associate such as amyloid-forming

peptides would be attractive candidates for the generation

of self-assembled structures with varying morphologies by

appropriate choice of surfaces and solvents for dissolution.

# 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90:

783–791, 2008.

Keywords: amyloid fibrils; beta structure; helical confor-

mation; peptide self-association; synthetic peptide

VVC 2008 Wiley Periodicals, Inc.

PeptideScience Volume 90 / Number 6 783

DWSFYLLYYTEFT showed greater enhancement of ThT flu-

orescence when compared with the longer peptide.7 Stock

solutions of amyloid-forming peptides are often prepared in

organic solvents such as dimethylsulfoxide (DMSO), trifluor-

oethanol (TFE), and hexafluoroisopropanol (HFIP) because

of their limited solubility in aqueous buffers.32–34 The sol-

vents TFE and HFIP play an important role in modulating

peptide conformation.35,36 They also facilitate dissolution of

peptide aggregates.32–34 We have examined the self-assembly

of Ac-DWSFYLLYYTEFT-am (denoted as Pb2m) when trans-

ferred from organic solvents to aqueous medium and mica

surface by ThT fluorescence and atomic force microscopy

(AFM), respectively.

MATERIALS AND METHODSFmoc amino acids were purchased from Novabiochem AG (Switzer-

land) and Advanced ChemTech (Louisville, KY). Peptide synthesis

resin, NovaSyn KR 125 was purchased from Novabiochem (La Jolla,

CA). All other reagents were of highest grade available.

Peptide SynthesisPb2m (Ac–DWSFYLLYYTEFT–am) was synthesized using standard

Fmoc chemistry.37 The synthesized peptide was cleaved from the

resin and deprotected using a mixture containing 82.5% trifluoro-

acetic acid, 5% phenol, 5% H2O, 5% thioanisole, and 2.5% ethane-

dithiol for 12 h at room temperature.38 The peptide was precipitated

in ice-cold diethyl ether. Peptide was dissolved in DMSO and puri-

fied on Hewlett Packard 1100 series HPLC instrument on a reversed

phase C18 Bio-Rad column. Purified peptide was characterized

using MALDI-TOF mass spectrometry on a Voyager DE STR mass

spectrometer (PerSeptive Biosystems, Foster city, CA) at the Proteo-

mics Facility in Centre for Cellular and Molecular Biology, India.

The m/z value observed was 1810.94 (calculated value: 1788.94),

which corresponds to the sodium adduct. Peptide stock solutions

were prepared in four different solvents: DMSO, MeOH, TFE, and

HFIP. The concentrations of the peptide were calculated using a

molar absorption coefficient of 9550M�1cm�1 at k ¼ 280 nm.

ThT FluorescenceThT fluorescence assay was performed using a modification of the

method described by Naiki et al.39 Phosphate buffer (50 mM, pH

7.0) was prepared in water. ThT (10 lM) in 50 mM phosphate

buffer, pH 7.0 was titrated with Pb2m stock solutions in four differ-

ent solvents: MeOH, TFE, HFIP, and DMSO. Pb2m concentration

was 1.7, 1.1, 1.7, and 1.7 mM in MeOH, TFE, HFIP, and DMSO,

respectively. The concentration of organic solvents was less than 5%

in aqueous phosphate buffer after dilution. Fluorescence spectra

were recorded on Fluorolog-3 model FL3-22 spectrofluorometer

(Horiba Jobin Yvon, Park Avenue Edison, NJ). The excitation wave-

length was set at 450 nm, slit width ¼ 2 nm and emission slit width

was set at 5 nm.

Circular DichroismCircular dichroism (CD) spectra were recorded on Jasco J-715 spec-

tropolarimeter. Far-UV spectra of 50 lM Pb2m were recorded in

MeOH, TFE, and HFIP. Spectra were recorded immediately after

dissolution of the peptides in the solvents and after 24, 48, and 96 h.

CD spectra were also recorded in 50 mM phosphate buffer, pH 7

immediately after diluting from the MeOH, TFE, and HFIP stock

solutions. The concentration of organic solvents was less than 5% in

aqueous phosphate buffer after dilution. In TFE and HFIP, spectra

were recorded from 250 to 180 nm. In MeOH and aqueous phos-

phate buffer, spectra could not be recorded at wavelengths less than

195 nm because of high absorbance of MeOH and buffer at lower

wavelengths.

All the spectra were recorded in 0.1 cm path length cell using a

step of 0.2 nm, band width of 1 nm and scan rate of 100 nm/min.

The spectra were recorded by averaging 10 scans and corrected by

subtracting the solvent/buffer spectra. Mean residue ellipticity was

calculated using following formula: [h]MRE ¼ (Mr 3 hobs)/(100 3l 3 c), where Mr ¼ mean residue weight, hobs ¼ ellipticity in milli-

degrees, l ¼ path length in decimeter, and c ¼ Pb2m concentration

in mg/ml.

Secondary structural components were estimated using CDSSTR

program available in CDPro software package.40,41 Pb2m spectra in

MeOH was deconvoluted for data range of 195–240 nm using

SMP56 protein reference set. Spectra recorded in TFE and HFIP

were deconvoluted for data range of 185–240 nm using SMP50 pro-

tein reference set.41

Atomic Force MicroscopyFor AFM studies, all the Pb2m samples in organic solvents (MeOH,

TFE, and HFIP) were deposited onto the freshly peeled mica surfa-

ces and allowed to air dry. Fresh 50 lM Pb2m solutions were also

prepared in MeOH, TFE, and HFIP for AFM imaging. Images were

acquired using tapping mode AFM (Multimode, Digital Instru-

ments, Santa Barbara, CA). A silicon nitride probe was oscillated at

275–310 KHz and images were collected at an optimized scan rate.

Analysis was done using Nanoscope III 5.30 r1.

Fourier Transform Infrared SpectroscopyFTIR spectra were recorded on a Bruker Alpha-E spectrometer with

Eco attenuated total reflection (ATR) single reflection ATR sampling

module equipped with ZnSe ATR crystal. Peptide samples at 50 lMand 0.7 mM were prepared in MeOH, TFE, and HFIP and the solu-

tions were kept at room temperature. TFA counterions were not

exchanged. Peptide samples were spread out and dried as films on

ZnSe crystal from each solvent. Each spectrum is the average of 24

FTIR spectra at a resolution of 4 cm�1.

RESULTS

ThT Fluorescence

Formation of amyloid fibrils by Pb2m when transferred to

aqueous buffer from organic solvents DMSO (1.7 mM),

MeOH (1.7 mM), TFE (1.1 mM), and HFIP (1.7 mM) at

784 Chaudhary, Singh, and Nagaraj

Biopolymers (Peptide Science)

concentrations indicated in parenthesis, was examined by

monitoring ThT fluorescence. Changes in fluorescence inten-

sity at 490 nm as a function of peptide concentration are

shown in Figure 1. The rise in fluorescence with increasing

peptide concentration indicates formation of amyloid fibrils.

Fluorescence increase is nonlinear and a second degree poly-

nomial was an appropriate fit for all the four curves. Maxi-

mum increase was observed when peptide was transferred

from TFE to buffer followed by transfer from HFIP, MeOH,

and DMSO solutions, respectively. Pb2m has been shown to

form fibrils rapidly (<2 min) when diluted from DMSO sol-

utions.7 Our results indicate that fibril formation takes place

rapidly when diluted from the organic solvents MeOH, TFE,

and HFIP.

CD Spectroscopy

Far-UV CD spectra were recorded in MeOH, TFE, HFIP and

in 50 mM PO43� buffer, pH 7.0 (see Figure 2). Panels A–C

represent the CD spectra of Pb2m in MeOH, TFE, and HFIP,

respectively, after incubation for 96 h at room temperature.

CD spectra were also recorded immediately, 24 and 48 h after

dissolution of the peptides in the three organic solvents. The

spectra of the peptide did not exhibit time-dependent

changes. Deconvolution of the spectra shown in Figures 2A–

2C using CDSSTR program indicated that the helix content

was 73.7%, 78.2%, and 74.8% and beta sheet content was

11.1%, 9.6%, and 8.4% in MeOH, TFE, and HFIP, respec-

tively. The analysis indicates that Pb2m adopts predomi-

nantly helical conformation in the three organic solvents.

Panels D–F show the CD spectra of Pb2m in 50 mM PO43�

buffer, pH 7.0 diluted from MeOH, TFE, and HFIP solutions,

FIGURE 1 Titration of 10 lM ThT in 50 mM phosphate buffer,

pH 7.0 with Pb2m stock solutions (1.7 mM in DMSO, 1.7 mM in

MeOH, 1.1 mM in TFE, and 1.7 mM in HFIP). Fluorescence emis-

sion at 490 nm was plotted against peptide concentration and data

could be fit to second degree polynomials.

FIGURE 2 Far-UV circular dichroism spectra of 50 lM Pb2m. Top panels show spectra in or-

ganic solvents after 96 h of incubation at room temperature. (A) MeOH; (B) TFE; (C) HFIP. Bot-

tom panels show spectra in 50 mM phosphate buffer, pH 7 immediately after diluting from MeOH

(D), TFE (E), and HFIP (F) stock solutions.

Self-Association of Amyloid Forming Peptide from �2-Microglobulin 785


respectively. When diluted into phosphate buffer, the spectra

show minimum *216 nm, characteristic of b-structure.Although the concentrations of the peptide are same, greater

negative ellipticity is observed when diluted from TFE and

HFIP solutions when compared with MeOH.

Atomic Force Microscopy

Figure 3 shows AFM image of Pb2m in 50 mM phosphate

buffer diluted from DMSO solution, incubated at 378C for

15 h and deposited on freshly peeled mica surface, washed

with deionized water and imaged after air drying. Extensive

fibril growth is observed as reported earlier.7 Under the con-

ditions of incubation, curved and modular morphology is

observed similar to full length b2m. AFM images of Pb2m,

after deposition and drying on mica, from stock solutions in

MeOH (1.7 mM), TFE (1.1 mM), and HFIP (1.7 mM) are

shown in Figure 4. Panel A indicates amorphous aggregates

for the MeOH sample. Similar amorphous aggregates were

also observed from HFIP (data not shown). When Pb2m was

deposited on mica from TFE, distinctive ring like structures

are clearly seen (panels B–E). The heights indicate that the

FIGURE 3 AFM imaging of 168 lM Pb2m in 50 mM phosphate

buffer, pH 7 diluted from 0.8 mM stock solution in DMSO and

incubated at 378C for 15 h. Scale bar represents 1 lm.

FIGURE 4 AFM imaging of Pb2m from stock solutions made in organic solvents. (A) MeOH,

1.7 mM and (B–E) TFE, 1.1 mM. Panel D represents the three-dimensional view of panel C. Panels

A–D represent the imaging of peptides on mica from undiluted Pb2m stock solutions while panel

E represents the imaging of peptide 20-fold diluted in TFE from stock solution. Scale bars represent

1 lm.



structures are in fact short tubes with a hollow interior and

length to diameter aspect ratios less than 1. Panels B–D are

images of Pb2m in TFE that was deposited on mica without

any dilution. Tubes of lengths ranging from 10 nm to more

than 50 nm are observed. Although, there is significant varia-

tion in length, the longest tubes are *50–60 nm in length.

AFM imaging does not give as good resolution in x-y direc-

tion as in z-direction and lateral resolution depends on tip

morphology.42 Therefore, it is not possible to accurately

measure the diameters of these tubes but their external diam-

eters are *200–400 nm. Panel E shows imaging of Pb2maggregates when stock solution was diluted 20 fold in TFE

before deposition on mica. The tube morphology is severely

affected and tube length in general is �30 nm, suggesting

that the tubular structures formed by Pb2m are not very rigid

and stable. However, these tubes give an insight into the

mechanism of tube formation by Pb2m in TFE. The rings

seem to be made up of various modules of small rod shape

aggregates self-assembling laterally to give tubular structures.

Self-association of Pb2m at a much lower concentration

of 50 lM in all the three alcohols was monitored after differ-

ent incubation periods by AFM. In MeOH (see Figure 5), the

major population is that of very small globular aggregates

with very few fibrillar aggregates. But with increasing time of

FIGURE 5 AFM imaging of 50 lM Pb2m in MeOH incubated at room temperature for 4 days.

Aliquots were removed at different time points and samples prepared for AFM imaging. Panels A,

B, and C represent the images recorded immediately after dissolution, 48 and 96 h of incubation,

respectively. Scale bars represent 1 lm.

FIGURE 6 AFM imaging of 50 lM Pb2m in TFE incubated at room temperature for 4 days. Ali-

quots were removed at different time points and samples prepared for AFM imaging. Panels A–B,

C and D–E represent the images recorded immediately after dissolution, 48 and 96 h of incubation,

respectively. Scale bars represent 1 lm.



incubation, a thicker population of elongated aggregates is

observed. In TFE (see Figure 6), aggregates at early time

points have a fibrillar morphology wherein the fibrils have

tapering ends (panels A, B). With time, these fibrillar species

seem to circularize to give the tubular aggregates (panels C–

E), thus suggesting an alternative mechanism of tube forma-

tion apart from the lateral association of rod like aggregates

as observed at higher Pb2m concentrations (Figure 4E).

Pb2m shows small globular aggregates in HFIP even at early

time points (Figures 7A–7C) but with increasing time, forms

fibrillar aggregates with tapering ends which circularize to

give rings/tubes which are quite different from those

observed in TFE (panels D, E). These tubes are *15–20 nm

in length, and unlike the tubes obtained in TFE, these tubes

have thinner walls and larger (*2 fold) inner diameter.

Apart from the tubes, a large population of globular aggre-

gates (*10–40 nm) is also present (panels D-E). We observe

that even at a low concentration of 50 lM, Pb2m has the abil-

ity to form aggregates when dried on mica from all the three

alcohols even after short incubation periods. To examine the

conformation of Pb2m aggregates in the solid-state, FTIR

spectra of Pb2m were recorded after drying from MeOH,

TFE, and HFIP at 50 lM and 0.7 mM concentrations.

Fourier Transform Infrared Spectroscopy

FTIR spectra of Pb2m in the amide I region, which is sensi-

tive to secondary structure,43,44 were examined. FTIR spectra

of Pb2m, dried from MeOH, TFE, and HFIP are shown in

Figure 8. Samples were prepared from stock solutions in

which peptide concentrations were 50 lM and 0.7 mM. Spec-

tra were recorded 4 days after dissolution of the peptide. The

spectrum for the TFE sample at 50 lM was also recorded af-

ter 5 days. Samples prepared from MeOH solutions showed

peaks at 1627 cm�1 (Figures 8A and 8B), which is character-

istic of amide I band of peptides adopting b-structure.43 Thepeak positions in the spectra from TFE solutions at 1628

cm�1 and 1653 cm�1 (corresponding to amide I band of

peptides in helical conformation43) indicate that b-structureis formed when dried from dilute solutions whereas a-helicalconformation predominates when dried form 0.7 mM solu-

tion (Figures 8C and 8D). When samples were prepared from

HFIP, peaks at 1653 cm�1 (Figures 8E and 8F) indicate heli-

cal conformation irrespective of the concentration of the

stock solution. Trifluoroacetate counterions were not

exchanged in any of the samples prepared for obtaining FTIR

spectra. Trifluoroacetate gives an absorption band around

1673 cm�1.45 A low intensity shoulder at *1673 cm�1 is

present in the spectra recorded on films dried from 50 lMPb2m solutions which can be attributed to trifluoroacetate

counterions. The intensity of the trifluoroacetate band is

diminished in the spectra of films obtained from 0.7 mM

Pb2m samples (Figures 8B, 8D, and 8F). The intensity and

position of the band because of trifluoroacetate does not

interfere with the assignment of the amide I band at 1653 cm�1

to helical conformation in the present study.

FIGURE 7 AFM imaging of 50 lM Pb2m in HFIP incubated at room temperature for 4 days.

Aliquots were removed at different time points and samples prepared for AFM imaging. Panels A,

B–C and D–E represent the images recorded after immediately after dissolution, 48 and 96 h of

incubation, respectively. Scale bars represent 1 lm.



The FTIR spectra recorded after 4 days of incubation in

MeOH and HFIP were identical to 1 day old samples except

when the peptide was dried from 50 lM TFE. Peaks for the

sample from TFE were observed at 1646 cm�1, 1635 cm�1,

and 1628 cm�1 for 1, 4, and 5-day-old samples. The peak at

1646 cm�1 indicates unordered structure44 at early time

points and b-structure after incubation for 5 days.

DISCUSSIONOur studies indicate that the amyloidogenic peptide span-

ning the b-strand E of wild-type human b2m can form amy-

loid fibrils when diluted from MeOH and helical-structure

promoting solvents such as TFE and HFIP into aqueous

buffer. In aqueous buffer, the peptide adopts b conformation,

although in organic solvents, helical conformation predomi-

nates. The fibril formation in buffer is rapid and is similar to

the observation when the peptide was diluted from DMSO7

in which it would be unstructured. Irrespective of the struc-

ture or lack of it in organic solvents, the peptide Pb2m adopts

b structure in aqueous medium and forms fibrils. Solvent de-

pendent self-assembled structures have also been observed

for a nine-residue peptide GAV-9.46 Effects of increasing

concentrations of HFIP and TFE on a monomeric peptide

spanning residues 20–41 of b2m indicated the ability of the

solvents to promote fibril formation.16 While helical confor-

mation was observed initially in the organic solvent-aqueous

mixtures, b-structure was observed on prolonged incubation

in the solvent mixtures. Maximum fibril formation was

observed at 20% TFE and 10% HFIP.16 Changes in morphol-

ogy from fibrillar to donut-like structure was observed for

insulin with increasing concentration of ethanol in aqueous

buffer.47 The presence of organic solvent in water would

result in decreased hydrophobic interactions between peptide

molecules. In neat fluorinated organic solvents such as TFE

and HFIP, the hydration shell would be replaced by alcohol

molecules followed by hydrogen bond and secondary struc-

ture formation such as helix or b-hairpin.35,36,48 We have

observed structures with varying morphologies when peptide

was dissolved in neat MeOH, TFE, and HFIP. In these sol-

vents, the peptide adopts predominantly helical conforma-

tion. The rapid evaporation of alcohols on the mica surface

would result in favorable hydrophobic interactions and inter-

actions between the adjacent aromatic residues resulting

in the formation of self-assembled structures other than

fibrils. The self-assembly process could occur in a manner

similar to the proposed mechanism for the formation of

nanotubes by phenylalanine dipeptides by Gazit and cow-

orkers49–52 and Phe containing peptide derived from Ab by

Krysmann et al.53,54 Although, no time-dependent changes

in secondary structure of Pb2m were observed, the time-de-

pendent changes in morphology observed from TFE and

HFIP solutions suggest a degree of self-association even in

these solvents. Although Pb2m adopts predominantly helical

structure in solution, FTIR spectra indicate that samples

dried from MeOH adopt b-structure in the solid-state. Also,

samples from TFE show time-dependent changes in confor-

mation when prepared from a stock solution at low concen-

tration. Initially, random conformation is observed. After

5 days, the peak position at 1628 cm�1 is characteristic of

amide I band of peptides adopting b-structure. When the

sample was prepared from a concentrated stock, b-structurewas not observed. Hence, the ring-like structures shown in

Figures 4B–4D arise from self-association of helical struc-

tures rather than b-structures. In fact, when b-structures areformed as in the sample from 50 lM TFE, the ring-like struc-

FIGURE 8 FTIR spectra in the amide I region for Pb2m dried

from 50 lM (Left panels) and 0.7 mM (Right panels) stock solu-

tions. The peptide was dried on ZnSe crystal from MeOH (A, B);

TFE (C, D); and HFIP (E, F). Panel C represents the spectrum

recorded after 5 days of incubation at room temperature. All other

panels are for samples incubated for 4 days.



tures are less prominent as shown in Figures 6D and 6E.

Unordered structure in the solid-state gives rise to elongated

structures shown in Figures 6A and 6B. Characterization of

the self-assembled structures at a molecular level would

require detailed molecular dynamics simulations studies.

There are several reports indicating that surface interac-

tions with mica and highly oriented graphite promote aggre-

gation of Ab into amyloid fibrils with different morpholo-

gies.55–57 Kowalewski et al.55 have shown that aggregation of

Ab (1–42) is highly dependant on nature of the solid surface.

On hydrophilic mica, Ab forms small aggregates at low con-

centrations which have tendency to form linear aggregates at

higher concentration while on hydrophobic silica, Ab forms

sheet like structures oriented at 1208 likely to be dictated by

crystallographic symmetry of graphite. Zhu et al.58 studied

the aggregation of Immunoglobulin light chain variable do-

main, SMA on hydrophilic and hydrophobic surfaces.

Hydrophilic mica facilitates the fibril formation under condi-

tions that give predominantly amorphous aggregates in solu-

tion. Fibrils are not formed on hydrophobic and positively

charged surfaces under identical conditions. Losic et al.56

studied aggregation of Ab (1–40) on highly oriented pyro-

lytic graphite. Ab forms linear aggregates along the step edges

of graphite and forms amyloid like fibrils on prolonged incu-

bation. In all these studies, fibril growth or aggregates forma-

tion was monitored on the surface as a function of time. Our

studies indicate that deposition of an amyloid-forming pep-

tide like Pb2m on mica by rapid evaporation of the organic

solvent in which the peptide was dissolved also leads to self-

associated structures with varying morphologies.

Amyloidogenic proteins and peptides tend to form amy-

loid fibrils from aqueous buffers in a time-dependent man-

ner.1–19 We have observed different morphologies when

Pb2m has been incubated for varying time periods in organic

solvents. The aggregates obtained from organic solvents do

not show amyloid fibrillar morphology. Ring-like structures

are prominent when Pb2m was dissolved in helix-promoting

solvents TFE and HFIP. Analysis of the CD spectra of Pb2mindicated the presence of both helical and b-conformations.

It is likely that this feature favors self-assembly with varying

morphologies on the mica surface. Since short peptides often

exist in an ensemble of conformations, it should be possible

to generate self-assembled structures with varying morpholo-

gies by appropriate choice of surfaces and solvents for disso-

lution of peptides, particularly those having the property to

self-associate such as amyloid-forming peptides.

We thank Kiran Kumar in Dr. Tushar Chakraborty’s laboratory in

the Indian Institute of Chemical Technology, Hyderabad, for help in

recording FTIR spectra.

REFERENCES1. Tjernberg, L. O.; Callaway, D. J.; Tjernberg, A.; Hahne, S.; Lillie-

hook, C.; Terenius, L.; Thyberg, J.; Nordstedt, C. J Biol Chem

1999, 274, 12619–12625.

2. Balbach, J. J.; Ishii, Y.; Antzutkin, O. N.; Leapman, R. D.; Rizzo,

N. W.; Dyda, F.; Reed, J.; Tycko, R. Biochemistry 2000, 39,

13748–13759.

3. Balbirnie, M.; Grothe, R.; Eisenberg, D. S. Proc Natl Acad Sci

USA 2001, 98, 2375–2380.

4. Zurdo, J.; Guijarro, J. I.; Jimenez, J. L.; Saibil, H. R.; Dobson,

C. M. J Mol Biol 2001, 311, 325–340.

5. Cottingham, M. G.; Hollinshead, M. S.; Vaux, D. J. Biochemis-

try 2002, 41, 13539–13547.

6. Reches, M.; Porat, Y.; Gazit, E. J Biol Chem 2002, 277, 35475–

35480.

7. Jones, S.; Manning, J.; Kad, N. M.; Radford, S. E. J Mol Biol

2003, 325, 249–257.

8. Nilsson, M. R.; Dobson, C. M. Biochemistry 2003, 42, 375–

382.

9. Zanuy, D.; Ma, B.; Nussinov, R. Biophys J 2003, 84, 1884–1894.

10. Goux, W. J.; Kopplin, L.; Nguyen, A. D.; Leak, K.; Rutkofsky,

M.; Shanmuganandam, V. D.; Sharma, D.; Inouye, H.; Kirsch-

ner, D. A. J Biol Chem 2004, 279, 26868–26875.

11. Frare, E.; Polverino De Laureto, P.; Zurdo, J.; Dobson, C. M.;

Fontana, A. J Mol Biol 2004, 340, 1153–1165.

12. Petty, S. A.; Adalsteinsson, T.; Decatur, S. M. Biochemistry

2005, 44, 4720–4726.

13. Wadai, H.; Yamaguchi, K.; Takahashi, S.; Kanno, T.; Kawai, T.;

Naiki, H.; Goto, Y. Biochemistry 2005, 44, 157–164.

14. Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel,

C.; Grothe, R.; Eisenberg, D. Nature 2005, 435, 773–778.

15. Rojas Quijano, F. A.; Morrow, D.; Wise, B. M.; Brancia, F. L.;

Goux, W. J. Biochemistry 2006, 45, 4638–4652.

16. Yamaguchi, K.; Naiki, H.; Goto, Y. J Mol Biol 2006, 363, 279–

288.

17. Ivanova, M. I.; Thompson, M. J.; Eisenberg, D. Proc Natl Acad

Sci USA 2006, 103, 4079–4082.

18. Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.;

Sievers, S. A.; Apostol, M. I.; Thompson, M. J.; Balbirnie, M.;

Wiltzius, J. J.; McFarlane, H. T.; Madsen, A. O.; Riekel, C.;

Eisenberg, D. Nature 2007, 447, 453–457.

19. Hamley, I. W. Angew Chem Int Ed Engl 2007, 46, 8128–8147.

20. Lopez De La Paz, M.; Goldie, K.; Zurdo, J.; Lacroix, E.; Dobson,

C. M.; Hoenger, A.; Serrano, L. Proc Natl Acad Sci USA 2002,

99, 16052–16057.

21. West, M. W.; Wang, W.; Patterson, J.; Mancias, J. D.; Beasley, J.

R.; Hecht, M. H. Proc Natl Acad Sci USA 1999, 96, 11211–

11216.

22. Sunde, M.; Blake, C. Adv Protein Chem 1997, 50, 123–159.

23. Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M.

B.; Blake, C. C. J Mol Biol 1997, 273, 729–739.

24. Gejyo, F.; Homma, N.; Suzuki, Y.; Arakawa, M. N Engl J Med

1986, 314, 585–586.

25. Kad, N. M.; Thomson, N. H.; Smith, D. P.; Smith, D. A.; Rad-

ford, S. E. J Mol Biol 2001, 313, 559–571.

26. Kad, N. M.; Myers, S. L.; Smith, D. P.; Smith, D. A.; Radford, S.

E.; Thomson, N. H. J Mol Biol 2003, 330, 785–797.

27. Smith, D. P.; Radford, S. E. Protein Sci 2001, 10, 1775–1784.



28. Chiti, F.; De Lorenzi, E.; Grossi, S.; Mangione, P.; Giorgetti, S.;

Caccialanza, G.; Dobson, C. M.; Merlini, G.; Ramponi, G.; Bel-

lotti, V. J Biol Chem 2001, 276, 46714–46721.

29. Bellotti, V.; Stoppini, M.; Mangione, P.; Sunde, M.; Robinson, C.;

Asti, L.; Brancaccio, D.; Ferri, G. Eur J Biochem 1998, 258, 61–67.

30. Jahn, T. R.; Parker, M. J.; Homans, S. W.; Radford, S. E. Nat

Struct Mol Biol 2006, 13, 195–201.

31. Gosal, W. S.; Morten, I. J.; Hewitt, E. W.; Smith, D. A.; Thom-

son, N. H.; Radford, S. E. J Mol Biol 2005, 351, 850–864.

32. Hirota-Nakaoka, N.; Hasegawa, K.; Naiki, H.; Goto, Y. J Bio-

chem 2003, 134, 159–164.

33. Zhang, H.; Kaneko, K.; Nguyen, J. T.; Livshits, T. L.; Baldwin,

M. A.; Cohen, F. E.; James, T. L.; Prusiner, S. B. J Mol Biol 1995,

250, 514–526.

34. Wood, S. J.; Maleeff, B.; Hart, T.; Wetzel, R. J Mol Biol 1996,

256, 870–877.

35. Rajan, R.; Balaram, P. Int J Pept Protein Res 1996, 48, 328–336.

36. Buck, M. Q Rev Biophys 1998, 31, 297–355.

37. Atherton, E.; Sheppard R.C. Solid Phase Synthesis: A Practical

Approach; Oxford: IRL Press, 1989.

38. King, D. S.; Fields, C. G.; Fields, G. B. Int J Pept Protein Res

1990, 36, 255–266.

39. Naiki, H.; Higuchi, K.; Hosokawa, M.; Takeda, T. Anal Biochem

1989, 177, 244–249.

40. Sreerama, N.; Woody, R. W. Anal Biochem 2000, 287, 252–260.

41. Sreerama, N.; Woody, R. W. Protein Sci 2004, 13, 100–112.

42. Kowalski, C.; Burrows, L.; Fitzgerald, C.; Thornton, J. Scanning

Probe Microscopy Training Notebook, Digital Instruments,

Veeco Metrology Group, 2003.

43. Haris, P. I.; Chapman, D. Biopolymers 1995, 37, 251–263.

44. Pelton, J. T.; McLean, L. R. Anal Biochem 2000, 277, 167–176.

45. Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry

1993, 32, 389–394.

46. Li, H.; Zhang, F.; Zhang, Y.; He, J.; Hu, J. Acta Biochim Biophys

Sin (Shanghai) 2007, 39, 285–289.

47. Dzwolak, W.; Grudzielanek, S.; Smirnovas, V.; Ravindra, R.;

Nicolini, C.; Jansen, R.; Loksztejn, A.; Porowski, S.; Winter, R.

Biochemistry 2005, 44, 8948–8958.

48. Blanco, F. J.; Jimenez, M. A.; Pineda, A.; Rico, M.; Santoro, J.;

Nieto, J. L. Biochemistry 1994, 33, 6004–6014.

49. Reches, M.; Gazit, E. Science 2003, 300, 625–627.

50. Reches, M.; Gazit, E. Nat Nanotechnol 2006, 1, 195–200.

51. Carny, O.; Shalev, D. E.; Gazit, E. Nano Lett 2006, 8, 1594–1597.

52. Reches, M.; Gazit, E. Phys Biol 2006, 3, S10–S19.

53. Krysmann, M. J.; Castelletto, V.; McKendrick, J. E.; Clifton, L.

A.; Hamley I. W.; Harris, P. J.; King, S. M. Langmuir 2008, 24,

8158–8162.

54. Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.;

Hule, R. A.; Pochan, D. J. Biochemistry 2008, 47, 4597–4605.

55. Kowalewski, T.; Holtzman, D. M. Proc Natl Acad Sci USA 1999,

96, 3688–3693.

56. Losic, D.; Martin, L. L.; Aguilar, M. I.; Small, D. H. Biopolymers

2006, 84, 519–526.

57. Wang, Z.; Zhou, C.; Wang, C.; Wan, L.; Fang, X.; Bai, C. Ultra-

microscopy 2003, 97, 73–79.

58. Zhu, M.; Souillac, P. O.; Ionescu-Zanetti, C.; Carter, S. A.; Fink,

A. L. J Biol Chem 2002, 277, 50914–50922.



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