Báo cáo khoa học: Tetracysteine-tagged prion protein allows discrimination between the native and converted forms pptx

Conformational dependence of the fluorescence of the FlAsH–TC-tagged mPrPs The fluorescence of the FlAsH reagent increased upon binding to the TC-tagged mPrP.. Addition of the native PrP

between the native and converted forms

Jernej Gasˇpersˇicˇ1, Iva Hafner-Bratkovicˇ1, Michel Stephan2, Peter Veranicˇ3, Mojca Bencˇina1,

Ina Vorberg4and Roman Jerala1,5

1 Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia

2 Department of Organic and Medicinal Chemistry, National Institute of Chemistry, Ljubljana, Slovenia

3 Faculty of Medicine, Institute of Cell Biology, Ljubljana, Slovenia

4 Institute of Virology, TU Munich, Germany

5 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia

Introduction

Prion diseases belong to a group of conformational

diseases characterized by the structural conversion of

native protein to alternative conformations [1] The

protein-only hypothesis states that prions are

com-posed predominantly of abnormally folded prion

pro-tein (PrP), the scrapie pathogenic form of PrP (PrPSc) [2] This form of PrP forms amyloid, which can be detected by compounds that bind to these types of ordered protein aggregate Molecules that bind specifi-cally to amyloids include thioflavin T (ThT) [3], Congo

Keywords

biarsenical; conversion; fibril; prion;

tetracysteine

Correspondence

R Jerala, Department of biotechnology,

National Institute of Chemistry, Hajdrihova

19, 1000 Ljubljana, Slovenia

Fax: +386 1 476 0300

Tel: +386 1 476 0335

E-mail: roman.jerala@ki.si

(Received 5 October 2009, revised 19

January 2010, accepted 17 February

2010)

doi:10.1111/j.1742-4658.2010.07619.x

The conformational conversion of prion protein (PrP) from a native con-formation to the amyloid form is a hallmark of transmissible spongiform encephalopathies Conversion is usually monitored by fluorescent dyes, which bind generic amyloids and are less suited for living cell imaging We report a new method for the synthesis of membrane-permeable and mem-brane-impermeable biarsenical reagents, which are then used to monitor murine PrP (mPrP) misfolding We introduced tetracysteine (TC) tags into three different positions of mPrP, which folded into a native-like structure Whereas mPrPs with a TC tag inserted at the N-terminus or C-terminus supported fibril formation, insertion into the helix 2–helix 3 loop inhibited conversion We devised a quantitative protease-free method to determine the fraction of converted PrP, based on the ability of the fluorescein arseni-cal helix binder reagent to differentiate between the monomeric and fibril-ized form of TC-tagged PrP, and showed that TC-tagged mPrP could be detected on transfected cells, thereby expanding the potential use of this method for the detection and study of conformational diseases

Structured digital abstract

l MINT-7709757 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by elec-tron microscopy ( MI:0040 )

l MINT-7709744 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by circular dichroism ( MI:0016 )

l MINT-7709730 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by fluor-escence technology ( MI:0051 )

Abbreviations

BME, b-mercaptoethanol; CrAsH, carboxy fluorescein arsenical helix binder; EDT, ethane dithiol; FlAsH, fluorescein arsenical helix binder; GPI, glycosylphosphatidylinositol; H, helix; mPrP, murine prion protein; PrP, prion protein; PrPSc,scrapie pathogenic form of prion protein;

TC, tetracysteine; TCEP, Tris(2-carboxyethyl)phosphine; ThT, thioflavin T.

red [4],

2-[1-(6-[(2-fluoroethyl)(methyl)amino]-2-naph-thyl)ethylidene]malononitrile [5], curcumin [6], and

luminescent conjugated polymers [7] Of these, ThT is

the most frequently used dye for detecting the

forma-tion of prion fibrils in vitro [8,9]

Localization of different proteins within living cells

became possible with the fusion of a fluorescent protein,

e.g green fluorescent protein, to the protein of interest

[10] Fluorescent protein domains may interfere with

protein folding or ligand binding and, more particularly,

with protein packing into ordered arrays Although the

green fluorescent protein fusion does not influence PrP

trafficking [11], it prevents prion infection [12], similarly

to the PrP–Fc fusion protein [13] A tetracysteine (TC)

tag that specifically binds biarsenic fluorescent dyes

rep-resents an alternative fluorescent labeling technique

A TC tag is a short peptide motif defined by the

sequence pattern CCXXCC, where C is a cysteine and

X any amino acid except cysteine [14] Fluorescein

arsenical helix binder (FlAsH) is an organoarsenical

compound that covalently binds to the TC tag at

subnanomolar concentrations [14] FlAsH is based on

fluorescein with two arsenic (III) atoms at the

4¢-posi-tion and 5¢-posi4¢-posi-tion, and is membrane-permeable

(Fig 1) This compound by itself is nonfluorescent [14]

Rigid spacing between both arsenic atoms in FlAsH

enables it to bind with high affinity and specificity to the

TC motif introduced into a variety of different proteins

[15] The binding of FlAsH reagent and also the

quan-tum yield of the formed FlAsH-peptide adduct depends

on the conformation of the peptide backbone adopted

by the tetracysteine tag [16] Tagging a protein with a

TC motif has been used for the detection of proteins

of interest in vivo [17,18], in purification [19], and for

detection on gel electrophoresis [15]

We decided to investigate the potential of

introduc-ing TC peptide tags at different positions on PrP in

order to monitor conformational changes in PrP We

show that insertion of a TC tag into the N-terminal and C-terminal segments of a protein does not inter-fere with fibril formation Fluorescent biarsenical reagents do not label the converted forms of PrP, but rather the native and denatured forms This property

is at the core of our new quantitative fluorescent con-version assay for PrP

Results Synthesis of FlAsH and carboxy FlAsH (CrAsH) The fluorescent reagents FlAsH and CrAsH, which bind to the TC peptide tag, have been previously reported on We modified the synthetic method described by Griffin et al [20] (Fig 1) The new practi-cal procedure limits the amounts of toxic reagents needed, and reduces the formation of byproducts by minimizing the number of manipulation steps during work-up Thus, starting from 4¢,5¢-bis(trifluoroacetoxy-mercuri)fluorescein, analytically pure FlAsH-ethane dithiol (EDT)2 was obtained in 29% yield (> 99% purity) after trituration with a minimal amount of

CH2Cl2 In addition, following the simplified proce-dure and starting from an approximately 1 : 1 mixture

of 4¢,5¢-bis(trifluoroacetoxymercuri)-5-carboxy-fluores-cein and 4¢,5¢-bis(trifluoroacetoxymercuri)-6-carboxy-fluorescein [21], an analytically pure mixture of 5-CrAsH-EDT2 and 6-CrAsH-EDT2 was obtained in 21% yield (> 99% purity) Both reagents were active and bound effectively to peptides containing the TC tag, as described below

Production, secondary structure analysis and thermal stability of the TC tag-modified protein Insertion of even a small peptide tag could affect the ability of PrP to convert to PrPSc We wanted to

Fig 1 Scheme of improved method of

chemical synthesis of FlAsH and CrAsH

reagents NMP, N-methyl-pyrolidinone;

RT, room temperature.

investigate which positions within the tertiary

structure of PrP allow the introduction of a peptide

tag without compromising protein folding and

conversion into the fibrillar structure We selected

three positions: one at the disordered N-terminal

segment of PrP (TCN), another at the C-terminal

segment, which connects PrP to the

glycosylphos-phatidylinositol (GPI) anchor (TCC), and a third at

a position within the PrP structure between

helix (H)2 and H3 that is surface-exposed, provides

the geometry that would allow binding of the

biarsenical reagent via the thiol groups, and requires

the most conservative amino acid replacements

(TCL) (Fig 2A) All recombinant proteins were

produced in Escherichia coli in the form of inclusion

bodies, and successfully refolded into the native-like

conformation as judged by the far-UV CD spectra

Native murine PrP (mPrP) has a high content of

a-helical secondary structure, with characteristic

minima at 210 nm and 222 nm in the far-UV CD

spectrum The spectra of TCC, TCN and TCL

overlap the spectra of mPrP, indicating that the

secondary structure is conserved in all TC-tagged

mPrPs (Fig 2B, left) The thermal stabilities of

TC-tagged mPrPs determined by CD spectroscopy,

when we introduced a TC tag at the C-terminus

(64 ± 0.5C), at the N-terminus before octare-peats (67 ± 0.5C), or in the loop between H1 and H2 (63 ± 0.5 C), were not substantially different from the thermal stability of mPrP alone (65 ± 0.5C) (Fig 2B, right)

Conformational dependence of the fluorescence

of the FlAsH–TC-tagged mPrPs The fluorescence of the FlAsH reagent increased upon binding to the TC-tagged mPrP Addition of the native PrP without a TC tag did not cause an increase in FlAsH fluorescence, demonstrating that this increase is due to specific binding to the TC tag In prion disease, the native a-form of PrP is converted to oligomers and amyloid fibrils with a high b-sheet content [22,23] We investigated whether the introduction of a TC tag into PrP allows discrimination between the conformational states of PrP The fluorescence of the FlAsH–TC-tagged mPrP complex increased with increasing con-centration of urea for TCN (Fig 3A), TCL (Fig 3B), and TCC (Fig 3C), suggesting that the fluorescence of the protein–FlAsH adduct is stronger when it is in the denatured conformation This was in contrast to the measurements on the nonstructured TC-containing peptide, which retained approximately the same

121

Octarepeats TCN

23

A

B

230

TCC

α2

α1

α3

β3 β1

TCL

Fig 2 TC tag insertion does not signifi-cantly affect protein secondary structure or stability (A) Structural representation of TC tags inserted into mPrP based on the solu-tion structure (Protein Data Bank code: 1XYX [62]), with the unstructured domain represented by dots (B) Left: comparison of far-UV CD spectra of mPrP and its

TC-tagged counterparts shows that the secondary structure is conserved in TC-tagged mPrPs Right: the thermal stability of TC-tagged mPrPs demonstrates similar melting temperatures Scans were obtained at protein concentrations of 0.1 mgÆmL)1in MilliQ water, with a temperature scan rate of 1 CÆmin)1, monitored by the ellipticity at 215 nm.

fluorescence regardless of the concentration of the

dena-turing agent, ruling out the solvent effect of the

denatur-ing agent on the FlAsH fluorescence (Fig 3D)

Several studies have shown transient interactions

between the flexible N-terminus and globular domain

[24–26] and ordering of the N-terminal octarepeats

above pH 6.5 [27], so the increase in fluorescence of

the FlAsH–TCN adduct with increasing denaturant

concentration could result from the change in the local

chemical environment of the PrP As a negative

con-trol, FlAsH fluorescence was measured in the presence

of increasing concentrations of denaturants with

(Fig 3E) or without (Fig 3F) mPrP One possible

explanation for the increase in FlAsH fluorescence

with increasing concentration of denaturant might be

that FlAsH binds more efficiently to the denatured

PrP than to the folded protein and that the higher

flu-orescence occurs because of additional binding under denaturing conditions However, we obtained the same effect when FlAsH was initially bound to the protein under denaturing conditions, and a lower final concentration of denaturing agent was obtained by dilution (Fig 3A–C) This demonstrates that TC tags

on PrP and FlAsH represent a conformation-sensitive probe

A TC tag at the N-terminus or C-terminus of PrP does not prevent conversion

We investigated whether the TC tag interferes with PrP conversion Fibril formation under mildly denatur-ing conditions [28] was monitored with the amyloid-specific dye ThT We showed that TCC and TCN formed fibrils, whereas TCL, which also folded into a

A

B

C

D

E

F

Fig 3 FlAsH fluorescence of TC-tagged

mPrP depends on the conformational state

of the protein The fluorescence of TCN (A),

TCL (B) and TCC (C) in the presence of

FlAsH was measured as a function of

con-centration of urea (s) In parallel, conjugates

of PrPs with FlAsH were formed in 8 M

urea, and diluted to the final concentration

of urea as indicated ( ) The fluorescence of

FlAsH bound to the short TC peptide

DDCCPGCCDD did not depend on the

pres-ence of denaturant (D) Control reactions

with mPrP without TC tag in the presence

of FlAsH (E) and FlAsH itself (F) do not

exhibit fluorescence.

native-like conformation, did not form fibrils within

4 days (Fig 4A) mPrP, TCN and TCC changed into

the b-structured conformation, whereas TCL remained

in the a-monomeric conformation (Fig 4B) TCN and

TCC fibril formation were also confirmed by

transmis-sion electron microscopy (Fig 4C), which showed

fibrils similar in size and morphology to those of

wild-type mPrP

Detection of TC-tagged mPrPs in cell culture

We introduced TC-tagged and non-TC-tagged mPrP

into the PrP-deficient cell line HpL3-4 Introduced

mPrPs additionally harbored mutations L108M and

V111M, enabling recognition by antibody 3F4

Although both TCC (Fig 5Ab) and TCN (Fig 5Ac)

were expressed at the cell surface, we had difficulties in

obtaining specific labeling with FlAsH using previously

published protocols [16,20,29–33] However, with a

modified protocol by Taguchi [34], the specific labeling

of TCC (Fig 5Bb) and TCN (Fig 5Bc) with FlAsH was

achieved We also used the CrAsH, which is similar to

FlAsH but contains an additional charged group and is

not membrane-permeable, to label only surface-exposed

TCC (Fig 5Cb) and TCN (Fig 5Cc) [35] No surface

FlAsH or CrAsH labeling was observed in HpL3-4

transduced with PrP without a TC tag (Fig 5Ba,Ca)

FlAsH fluorescence discriminates native from

fibrillar mPrP

As we had demonstrated that TCC and TCN have the

ability to be converted into fibrils, we investigated

FlAsH fluorescence in combination with the native or fibrillar form of TC-tagged PrP FlAsH binding and its fluorescence was followed in parallel with ThT fluo-rescence during fibril formation We found that FlAsH selectively bound to the native forms of TCN and TCC and exhibited fluorescence In the presence of TCN and TCC fibrils, however, FlAsH exhibited no fluorescence (Fig 6A,C) The fluorescence of ThT was inversely proportional to the fluorescence of FlAsH When amyloid started to form, ThT fluorescence increased, and at the same time FlAsH fluorescence decreased along with the amount of monomeric PrP

In the case of TCL, which does not form fibrils, both ThT and FlAsH fluorescence remained unchanged (Fig 6B) This phenomenon could be explained in two ways: either FlAsH does not bind to the fibrillar form

of TCN and TCC, because the TC tag becomes inac-cessible to the reagent; or the cysteines of the TC tag become oxidized during fibril formation However, the addition of a reducing agent to PrP fibrils did not result in enhanced FlAsH fluorescence intensity (data not shown), and the addition of FlAsH to nonconvert-ing TCL resulted in fluorescence under the same reac-tion condireac-tions Thus, we conclude that the TC tag in fibrils becomes inaccessible to FlAsH This is also sup-ported by the finding that fibrils made from TCC pre-labeled with FlAsH under native conditions retained their fluorescence (Fig 6D)

Quantitative fluorescent PrP conversion assay

We showed that TC-tagged PrP and FlAsH represent

a sensor for PrP conversion This phenomenon could

A

C

B

Fig 4 Conversion of TC-tagged PrPs (A) The kinetics of fibril formation of mPrP ( ), TCC ( ), TCN (•) and TCL (.) in microtiter plate assays were monitored by the fluores-cence of ThT TCC and TCN formed amy-loid, whereas TCL did not undergo conversion (B) CD spectra of converted PrP show that mPrP, TCC and TCN were converted to the b-form, whereas TCL remained in the a-conformation (C) Formation of PrP fibrils of TCC, TCN and mPrP was confirmed by transmission electron microscopy.

be used to quantify PrP conversion without the need

to perform proteolytic digestion of converted PrP,

which is the basic principle of most conversion assays

We have devised an assay to determine the fraction of

converted PrP, based on the difference in fluorescence

between the monomeric and fibrillar forms of TCC

FlAsH detects only the amount of nonconverted

TC-tagged PrP In order to obtain an accurate result

for the conversion efficiency, we needed to normalize

the reading of the nonconverted TC-tagged PrP

against the total amount of TC-tagged PrP in the

sam-ple This can be determined by solubilizing all, i.e

con-verted and nonconcon-verted, TC-tagged PrP into the

soluble denatured form by using 6 m guanidine

hydro-chloride and 10 mm dithiothreitol The denatured form

was fluorescent, owing to the binding of FlAsH, and

was used to determine the amount of total TC-tagged

PrP from the calibration curve for the unfolded

pro-tein (Fig 7C) We showed that mixtures of different

ratios of converted and native TC-tagged PrP showed

a linear response over the whole range (Fig 7D), thus providing a method for detecting TC-tagged PrP conversion without the need to employ proteolytic digestion

Discussion Different fluorescent tags have revolutionized protein science and cell biology However, large and slow-fold-ing fluorescent proteins may influence the foldslow-fold-ing of their fusion partner, and, in the case of prion disease and other conformational diseases, they may interfere with conformational conversion, as demonstrated for PrP [11,12] Therefore, small, genetically encoded tags may prove more useful in prion research, particularly with cell culture assays for testing prion infectivity [36,37]

We introduced a TC tag, enabling the selective incorporation of biarsenical fluorophores into different positions on PrP Ideally, tags should not affect

0 µm 25

0 µm 25

A

B

C

Fig 5 Fluorescent biarsenical compounds

specifically label TC-tagged PrPs expressed

in the PrP-deficient cell line HpL3-4 (A)

mPrP (a), TCC (b) or TCN (c) are expressed

at the cell surface, as judged from flow

cytometry analysis using 3F4 as primary

antibody (white) Cells stained only with

Cy2-conjugated secondary antibodies were

used as controls (gray) Cells were stained

with FlAsH (B) or CrAsH (C), and imaged

under the confocal microscope In cells that

express TCC (Bb and Cb) and TCN (Bc and

Cc), FlAsH and CrAsH selectively stain the

cell surface, whereas there is no staining of

the cells expressing mPrP (Ba and Ca)

protein.

protein stability or trafficking, and should not interfere

with prion misfolding We avoided introducing TC

tags into segments around the residues that had been

previously shown to contribute to the species barrier

[38] or that show a different susceptibility to prion

strains, with the aim of developing a test with broad

applicability None of the three introduced TC tags

substantially affected protein secondary structure

mPrPs with TC tags inserted at the N-terminus or

C-terminus were able to undergo conversion, whereas

introduction of a TC tag into the loop between H2

and H3 prevented fibril formation TCN and TCC

seem to undergo fibrillization even faster than mPrP,

and we are currently investigating the mechanism of

this phenomenon Although antibody studies [39] and

some PrPScmodels [40,41] do not predict that the

seg-ment in the loop between H2 and H3 will undergo a

conformational change, some human pathogenic

muta-tions, such as E196K [42], indicate that this segment

might influence PrP misfolding We speculate that this

segment forms contacts with other monomers in the

core of the fibrils, although PrP has been previously

shown to be sensitive to point mutations throughout

its compact domain

Biarsenical reagents have great potential as

molecu-lar sensors, particumolecu-larly as improved cell-staining

pro-cedures allow a high signal-to-background noise ratio

The new simplified synthetic procedure reported here

may allow researchers to prepare reagents to stain

either all cellular TC-tagged proteins or only a subset

exposed at the cell surface We used a modified cell-labeling method [34] to show that FlAsH and CrAsH specifically label TC-tagged PrPs CrAsH is particu-larly suitable for following the trafficking of PrPs and other TC-tagged extracellular proteins, as it does not cross the cell membrane, owing to the presence of charged groups, and its fluorescence is more stable at physiological pH [35]

FlAsH fluorescence increased with protein denatur-ation, showing that FlAsH in the context of PrP is a conformation-sensitive probe This is similar to what was found in a previous study, where FlAsH fluores-cence increased upon unfolding of TC-tagged cellular retinoic acid-binding protein I [16] On the other hand, under PrP conversion conditions, FlAsH fluorescence decreased with an increase in ThT fluorescence, which marks the formation of amyloid fibrils As the pre-sence of reducent did not reconstitute FlAsH binding

to fibrils (data not shown), the best explanation is that the TC tag in fibrils of TCN and TCC is not accessible

to FlAsH, and that both the N-terminal and C-termi-nal regions are buried within the formed fibrils This is

in agreement with the results of antibody-binding stud-ies [43,44] Additionally, a GPI anchor attached to the C-terminus of PrPSc cannot be cleaved by phospholi-pase C, indicating that this PrP segment is protected after formation of the protease-resistant form of PrP [45], which is in accordance with our results An MS study showed that the flexible N-terminus is highly protected in fibrils in comparison to native PrP [46]

0 µm 25

D C

Fig 6 FlAsH follows the conversion of TCC and TCN in an inversely proportional manner relative to ThT Structural conversion of TCN (A), TCL (B) and TCC (C) was followed by ThT ( ) and FlAsH fluorescence (s) Aliquots of the fibril formation mixture were taken at the indicated times Samples were incubated with FlAsH for 2 h at room tem-perature, with 1 m M TCEP, 1 m M BLE and

50 m M Hepes (pH 7.5) prior to fluorescence measurement The fluorescence intensity at emission maxima (kFlAsH= 528 nm,

k ThT = 474 nm) is plotted against conversion time The FlAsH–TCC adduct was prepared prior to fibril formation, and left to undergo conversion for 48 h Fluorescent aggregates were visualized by confocal microscopy (D).

Recently Coleman et al [47] observed an increase in

FlAsH fluorescence when it was bound to oligomeric

PrP as compared with a-structured PrP In their

con-struct, a TC tag was introduced into the hydrophobic

domain of PrP, N-terminally to the structured domain

This region has been shown to participate in the

amy-loid core [41,48–50], so it is possible that their result

was a consequence of observing b-oligomers, as the

presence of mature fibrils has not been demonstrated

We used the selectivity of FlAsH fluorescence

between the native and converted forms of TC-tagged

PrP as the basis of a new quantitative fluorescent PrP

conversion assay (Fig 7B) Most assays for PrP

conver-sion are based on the proteolytic degradation of PrP by

proteinase K, which is time-consuming and, above all, has to be carefully calibrated for the amount of the active protease and the duration and reaction condi-tions of digestion, which can significantly affect the result [36,51] In our assay, we can quantify the amount

of nonconverted TC-tagged PrP by measuring the fluo-rescence after the addition of FlAsH The total initial amount of TC-tagged PrP in the sample required for the normalization and comparison between different samples can be determined from the FlAsH fluores-cence of the solubilized and unfolded PrP (Fig 7A) The principle of our described assay is similar to that of the conformation-dependent immunoassay [52], where one set of antibodies is used to detect the amount of

23

23

A

D

Fig 7 Quantitative fluorescent PrP

conver-sion assay (A) Schematic illustration of the

principle of the assay: under native

condi-tions, only native PrP exhibits fluorescence

(indicated by a star), whereas after the

addition of denaturant, all forms of PrP are

solubilized and exhibit FlAsH fluorescence.

(B) Fluorescence emission spectra of the

native and converted form of TC-tagged PrP

(TCC) in the presence of FlAsH (C)

Fluores-cence of converted and nonconverted PrP in

the presence of 6 M guanidine hydrochloride

and 10 m M dithiothreitol (D) Samples with

different fractions of converted PrP were

prepared by mixing converted and native

TCC FlAsH fluorescence was determined

under native conditions, and normalized by

the fluorescence under the denaturing

con-ditions of each sample divided by the

fluo-rescence of native PrP under the denaturing

conditions, displaying a linear response.

nonconverted PrP, and the total amount of PrP,

unfolded in the presence of denaturing agent, is

deter-mined using the second set of antibodies The described

method could be particularly useful in research

requir-ing fast results, such as in kinetic and structural studies

of PrP intermediates, for in vitro conversion, such as in

an amyloid seeding assay [9] and protein misfolding

cyclic amplification [53,54], and potentially also in

diag-nostics in cell-based assays based on the reporter cell

line producing the TC-tagged PrP [55]

Experimental procedures

Materials

The 3F4 antibodies were purchased from Dako (Glostrup,

Denmark), Cy2-conjugated anti-mouse IgG from Dianova

(Hamburg, Germany), pRSET A plasmid from Invitrogen

(Madison, WI, USA), the Quikchange kit from Stratagene

(La Jolla, CA, USA), and Ni2+–nitrilotriacetic acid resin

from Qiagen (Hilden, Germany) Guanidine hydrochloride

and urea were purchased from Fluka (Buchs, Switzerland)

All other chemicals were purchased from Sigma (St Louis,

MO, USA)

Preparation of TC-tagged PrP constructs

TC tags were introduced into three positions on 3F4-tagged

murine PrP (L108M⁄ V111M) In TCC, CCPGCC was

inserted after Ser231 Mutations in TCL were T192C,

T193C, K194P, E196C, and N197C Mutations in TCN

were R37C, Y38C, Q41C, and G42C (Fig 2A)

Insertions were introduced into the 3F4-tagged mPrP

ORF (from 23 to 230 amino acids) cloned into plasmid

pRSET A (Invitrogen) with a Quikchange kit (Stratagene),

using specific sense and complementary antisense

oligonu-cleotides, as follows: for TCC sense, 5¢-GGG CGT CGT

TCC AGC TGT TGT CCG GGT TGT TGT TAA GAA

TTC GAA GC-3¢; for TCC antisense, 5¢-GC TTC GAA

TTC TTA ACA ACA ACC CGG ACA ACA GCT GGA

ACG ACG CCC-3¢; for TCN sense, 5¢-AAC ACC GGT

GGA AGC TGT TGT CCT GGT GTT GTA GCC CTG

GAG GCA AC-3¢; for TCN antisense, 5¢-GT TGC CTC

CAG GGC TAC AAC ACC AGG ACA ACA GCT TCC

ACC GGT GTT-3¢; for TCL sense, 5¢-CAC ACG GTC

ACC ACC TGC TGC CCG GGG TGC TGC TTC ACC

GAG ACC GAT-3¢; and for TCL antisense, 5¢-ATC

GGT CTC GGT GAA GCA GCA CCC CGG GCA GCA

GGT GGT GAC CGT GTG-3¢

Protein expression, purification, and refolding

Plasmid pRSET A, encoding mPrP (or TC-tagged mPrP:

TCN, TCL, or TCC), was transformed into competent

E coliBL21(DE3) pLysS, and mutated PrPs were expressed

in the form of inclusion bodies The protein was purified and refolded on an Ni2+–nitrilotriacetic acid column, using a similar procedure to one previously described [6,56] The pur-ity of the isolated protein was checked by SDS⁄ PAGE

Conversion to the fibrillar form of PrP

Proteins were denatured overnight in 6 m guanidine hydro-chloride at 4C The amyloid form of mPrP was produced

by diluting denatured mPrP and TC-tagged mPrP with 1 m guanidine hydrochloride, 3 m urea and NaCl⁄ Pi(pH 6.8) at protein concentrations of 20 lm, and shaking at 37C [28] The amyloid form of PrP in 96-well microtiter plates was produced by an identical protocol, and three 3⁄ 32 teflon balls were also added to each well for better shaking [57] FlAsH cannot bind to the formed fibrils; however, labeled PrP fibrils can be prepared by conversion of the FlAsH-labeled PrP (TCC), as the label does not hinder con-version FlAsH was added to native TCC at a 2 : 1 ratio,

in a reaction mixture containing 50 mm Hepes, 1 mm Tris(2-carboxyethyl)phosphine (TCEP), and 1 mm b-mer-captoethanol (BLE) The reaction solution was incubated

at room temperature for 2 h, protected from light Fibrils made from FlAsH-labeled TCC were made using an identi-cal protocol to the one described above

Electron microscopy

Holey formvar carbon-coated copper grids (SPI Supplies) were coated with 0.1% poly(l-lysine) and placed on one drop of the protein sample for 3 min Samples on grids were negatively stained with 1% (w⁄ v) aqueous uranyl ace-tate, and observed on a Jeol 100CX electron microscope operating at 80 keV

CD spectroscopy

CD spectra were recorded on an Applied Photophysics Chirascan spectropolarimeter under nitrogen flow Far-UV

CD spectra for protein secondary structure determination were recorded between 190 nm and 260 nm in a 0.1 cm pathlength cuvette at a protein concentration of 0.1 mgÆmL)1, using steps of 0.5 nm with 1 s per point The temperature stability of proteins was recorded in a 0.1 cm pathlength cuvette (300 lL) at a protein concentra-tion of 0.1 mgÆmL)1 with a temperature scan rate of

1CÆmin)1 at 215 nm Spectra were smoothed by software supplied with the instrument

Fluorescence spectroscopy

For fluorescence measurements, a Perkin Elmer LS55 fluo-rimeter was used ThT emission (460–535 nm) was tracked

by excitation at 442 nm with a protein concentration of

1 lm and 10 lm ThT

The fluorescence of FlAsH was monitored between

510 nm and 560 nm with excitation at 508 ± 5 nm in a

0.3 cm pathlength cuvette FlAsH (5 lm) was incubated at

room temperature for 2 h with 5 lm protein or peptide in

the presence of 1 mm BME and 1 mm TCEP in 50 mm

Hepes (pH 7.5) [16]

Conversion calculation

The amount of converted PrP was calculated from the

fluo-rescence intensity values of the sample after the 1 h

incuba-tion period in the presence of 10 lm FlAsH, 1 mm TCEP,

1 mm BME, and 50 mm Hepes (pH 7.5), and after reading

of the same sample after the addition of 6 m guanidine

hydrochloride, 10 mm dithiothreitol, 50 mm Hepes

(pH 7.5), and 10 lm FlAsH The amounts of native and

denatured PrP were read from the calibration curve

obtained with different amounts of PrP under native and

denaturing conditions in the presence of FlAsH, in both

cases with subtracted fluorescence of sample with FlAsH

without TC-tagged prion protein

The percentage of converted PrP was calculated using the

equation

fraction converted¼ ðPrPtot PrPnatÞ=PrPtot

where PrPnat represents the nonconverted amount and

PrPtot the total amount (converted plus nonconverted) of

PrP

Construction of retroviral plasmids, production of

retrovirions and transduction of the HpL3-4 cell

line, and flow cytometry

TC tags were introduced into the 3F4-tagged mPrP ORF in

pcDNA3.1zeo(+) by Quikchange mutagenesis (Stratagene)

Whereas the N-terminal TC tag was prepared using the

same oligonucleotides as given above, sense (5¢-TCC

CAG GCC TAT TAC TGT TGT CCA GGA TGT TGT

GAC GGG AGA AGA TCC-3¢) and antisense (5¢-GGA

TCT TCT CCC GTC ACA ACA TCC TGG ACA ACA

GTA ATA GGC CTG GGA-3¢) oligonucleotides were used

to insert the TC tag before the GPI attachment signal

Mutant mPrP ORFs were subcloned into the retroviral

expression vector pSFF [58–60] The pSFF vectors were

transfected into a coculture of packaging cell lines w2 and

PA317 When cells were more than 80% positive for PrP,

retroviral supernatants were harvested and cleared by

cen-trifugation (120 g, 4C, 10 min) HpL3-4 cells (3 · 105per

well) [61] were plated into six-well microtiter plates 1 day

before transduction Cells were incubated with polybrene

(4 lgÆmL)1) 2 h before addition of the retrovirions One

milliliter of retroviral supernatant was incubated with the

cells for 2 days, after which the cells were transferred to a

6 cm or 10 cm culture plate

We used a flow cytometry protocol adapted from Maas et al [37] to check whether TC-tagged proteins are expressed at the cell surface of HpL3-4 cells similarly to wild-type PrP Cells (5· 105

per tube) were first incubated with FACS buffer (2.5% fetal bovine serum in NaCl⁄ Pi) for 10 min at 4C One hundred microliters of 3F4 anti-body (5 lgÆmL)1; Dako) was added to the cells and incu-bated for 45 min at 4C After washing, the cells were incubated with Cy2-conjugated anti-mouse IgG as second-ary antibodies (Dianova) for 45 min at 4C in the dark Rinsed cells were analyzed by flow cytometry

Cell labeling with biarsenical compounds

Cells were stained with FlAsH (CrAsH) using an adapted protocol [34] Cells were incubated in l-Slide eight-well slides (iBidi) overnight Prior to staining, FlAsH (CrAsH) was preincubated with 0.8 m dithiothreitol and 5 mm EDT for 5 min at room temperature Cells were washed with HBSS and stained for 1 min with 1.3 lm FlAsH (CrAsH) and 15 mm dithiothreitol in HBSS Unbound FlAsH (CrAsH) was rinsed off with HBSS Cells were fixed either with 4% paraformaldehyde or ice cold ()20 C) methanol for at least 5 min

Confocal microscopy

Images were obtained on a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with an HCX plan apo ·63 oil (numerical aperture: 1.4) oil immersion objective For excitation, the 514 nm line of a 25 mW argon laser was used As laser power of 5% was used for the argon laser Fluorescence emission was detected at 530–560 nm

Acknowledgements

We would like to thank D Oven and R Rost for excellent technical assistance We are grateful to

A Aguzzi for the plasmids for mPrP expression,

S A Priola and B Chesebro for providing the w2 and PA317 cells and the vector pSFF, and T Onodera for the HpL3-4 cells We would like to thank C Taft for careful reading of the manuscript The authors acknowledge financial support from the state budget

by the Slovenian Research Agency This project was supported by the 6th framework EU project, TSEUR

References

1 Stefani M (2004) Protein misfolding and aggregation: new examples in medicine and biology of the dark