Fluorescent ratiometric MFC probe sensitive to early stages of α-Synuclein aggregation

Article abstract of DOI:10.1021/ja102838n

We introduce a sensor molecule, AS140-MFC, consisting of a covalent adduct of an Ala-to-Cys mutant of α-synuclein with the 3-hydroxychromone dual emission dye MFC. We show that the AS140-MFC construct is a multiparametric fluorescent probe suitable for the continuous monitoring of protein aggregation and is sensitive to the early and intermediate stages of α-synuclein aggregation, a process associated with Parkinsons disease.

Full text of DOI:10.1021/ja102838n

Published on Web 05/21/2010
Fluorescent Ratiometric MFC Probe Sensitive to Early Stages of r-Synuclein
Aggregation
Dmytro A. Yushchenko,† Jonathan A. Fauerbach,‡ Shyamala Thirunavukkuarasu,†
Elizabeth A. Jares-Erijman,‡ and Thomas M. Jovin*,†
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11,
37077 Go¨ttingen, Germany and Departamento de Qu´ımica Orga´nica, Facultad de Ciencias Exactas y Naturales,
UniVersidad de Buenos Aires, CIHIDECAR, CONICET, 1428 Buenos Aires, Argentina
Received April 5, 2010; E-mail: tjovin@gwdg.de
The aggregation of the 140aa protein R-synuclein (AS) is
associated with a prominent cytopathological manifestation of
Parkinson’s disease (PD), the deposition of dense tangles of fibrils
with a characteristic cross-ꢀ amyloid structure in dopaminergic
neurons of the substantia nigra of the midbrain.1 Extrinsic
fluorescent dyes such as thioflavin T (ThT) display changes in
intensity, spectral distribution, and polarization upon binding to
amyloid fibrils and are extensively used for their quantitation and
visualization.2 Unfortunately, aggregation assays generally require
periodic sampling of the inherently complex molecular populations,
leading to poor reproducibility and insufficient definition of the
reaction course. More importantly, most probes are insensitive to
Figure 1. (A) Scheme for the synthesis of the MFC probe. (B) Diagramatic
representation of AS140-MFC and its fluorescence excitation (blue) and
emission (red) spectra at pH 6.2 and 25 °C.
the early oligomeric states of AS aggregation presumed to mediate
the cytotoxic processes leading to the progression of PD.1c,3
Improved tools for monitoring AS aggregation in Vitro and in ViVo
are urgently required.
The steps used in the synthesis of MFC are shown in Figure 1A
and documented in the Supporting Information (SI). The particular
AS construct featured here and denoted AS140-MFC is a Cys
mutant of AS in position 140 (C-terminus) reacted with MFC. The
labeled protein AS140-MFC has two well-defined emission bands
(Figure 1B; SI) with a T*/N* ratio (1.1 at 25 °C) close to that
(1.2) for a reference compound, the conjugate of MFC with
acetylcysteine (SI). This finding is consistent with the classification
of AS as an intrinsically disordered protein.1
AS140-MFC is suitable for the continuous monitoring of protein
aggregation and, in contrast to ThT, is sensitive to the early and
intermediate stages of the overall reaction (Figure 2). We initiated
the aggregation of a mixture of WT AS and 2.5% AS140C-MFC
(150 µM total protein, pH 6.2) by exposure to 37 °C with vigorous
agitation and acquired emission spectra as a function of time. In
parallel, we sampled the reaction periodically with the ThT assay.
The time course of a number of parameters derived from the spectra
are featured in Figure 2A. The most notable spectroscopic feature
was a dramatic (15-fold) increase in the T* band intensity, with a
smaller, 2-fold increase of the N* band. The net effect was a 9.5-
fold rise in the T*/N* ratio to a peak value of 8.8 at 20 h, decreasing
to 7.6 at the end of the reaction (70 h).
Environment-sensitive dyes are particularly useful reporters of
protein aggregation.4 We have previously described the utility of
pyrene5a and aminonaphthalenes5b for monitoring the various stages
of AS aggregation. In addition, fluorescence correlation (FCS)6 and
energy transfer (FRET)6,7a techniques using conventional dyes6 and
expression probes7a provide “early reporting” and can be applied
in combination with functionalized nanoparticles in living cells.7b
The 3-hydroxychromones (3HCs) constitute an exceptional
family of fluorophores with unique environmental sensitivity. These
compounds exhibit a rapid (subns) excited-state intramolecular
3-hydroxyl f 4-carbonyl proton transfer (ESIPT), resulting in
distinct emissions from both the initial “normal” excited state (N*)
and the ESIPT phototautomer product (T*). The differential
response of the N* and T* bands to environmental changes is
conveniently expressed as the ratio of band intensities IT*/IN* (T*/
N* ratio) and other spectral relationships.8 A decrease in the polarity
and H-bonding (donor) capacity of the microenvironment favors
the ESIPT reaction of HCs, thereby increasing the relative intensity
of the T* band. These unique properties have led to the application
of HCs as membrane probes and as sensors of protein-DNA,
DNA-polycation, and protein-protein interactions.8,9
We have previously reported that some 3HCs reveal differences
in the supramolecular organization of amyloid fibrils formed by
wild type (WT) AS and disease-related familial mutants.10 In this
work we introduce sensor molecules consisting of covalent adducts
of Ala-to-Cys mutants of AS with a thiol-reactive (maleimide) probe
(MFC) based on 2-(2-furyl)-3-hydroxychromone FC9,11 (Figure 1).
They are representative of the new, versatile ESIPT probes of
amyloid aggregation, with unprecedented sensitivity for the initial
stages of reaction.
Particularly noteworthy was that these and other changes
preceded the progress curve defined by the conventional ThT signal
(Figure 2B). The sequence of t1/2 values (transition midpoints) were
as follows: T* peak wavelength (5.3 h), T* peak intensity (12.5
h), N* peak intensity (13.8 h), 355 nm scattering (23.5 h), and ThT
signal (34 ( 2 h). These values and other qualitative and
quantitative features of the progress curves (early bathochromic
shift of the T* band, intersection of T* and N* fractional transitions,
transient maximum of the T* band) constitute unambiguous
evidence for reaction intermediates (I) detected by the ESIPT probe
by virtue of differing significantly from the monomeric and amyloid
† Max Planck Institute for Biophysical Chemistry.
‡ Universidad de Buenos Aires.
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7860 J. AM. CHEM. SOC. 2010, 132, 7860–7861
10.1021/ja102838n  2010 American Chemical Society

C O M M U N I C A T I O N S
fibrillar (AF) forms of AS. Such transient pre-ꢀ (see below)
conformational switching has also been detected by the sophisticated
application of FCS and FRET.6 We visualized the intermediates
by AFM (an example is shown in the SI; a detailed description
will be supplied in a separate communication) and are currently
attempting their biochemical isolation, a difficult undertaking due
to their elusive nature.6 Light scattering at 355 nm largely paralleled
the ThT signal but judging from the lower t1/2 appeared to sense
some forms of I as well as AF.12
evidence5a for immobilization of virtually all protein segments on
the nanosecond time scale.
An important practical advantage of the MFC probe is the
inherently continuous nature of the aggregation assay, providing
more detailed, multiparameter data than the usual ThT-based
sampling protocols. The procedure can be implemented on many
instrumentation platforms and is easily automated. We anticipate
that AS140-MFC and related constructs will constitute useful tools
for screening inhibitors or reversers of AS aggregation, as well as
being readily transferable to other proteins undergoing pathological
as well as functional1a,15 amyloid transitions.
Acknowledgment. We thank Drs. A. Demchenko, V. Shvad-
chak, D. Arndt-Jovin, and R. Klement for valuable discussions and
U. Plessmann for mass spectra. Supported by the Max Planck
Society (E.J.E., Partner Group grant; Toxic Protein Conformation
project); Cluster of Excellence 171 of the DFG Centre for Molecular
Physiology of the Brain (DFG CMPB); Argentine agencies Anp-
CyT, CONICET, UBA (E.J.E.). DAY received postdoctoral fel-
lowships from EU FP6 STREP Fluoromag project 037465II and
the Marie Curie Foundation.
Supporting Information Available: Synthesis and characterization
of MFC dye. Preparation of AS and labeling procedure with MFC.
Spectral properties of AS and AFM images at the different steps of
aggregation. This material is available free of charge via the Internet
at http://pubs.acs.org.
Figure 2. Aggregation of 2.5% AS140-MFC + 97.5% WT AS (150 µM
total protein) at 37 °C. (A) Derived spectral signals, normalized by their
changes between the start and end of the incubation and plotted according
to their respective scales: T* peak wavelength (black curve), T* peak
intensity (red), N* peak intensity (blue). The N* peak wavelength (not
shown) did not vary significantly (433 ( 2 nm). Insert: repetitive emission
spectra during aggregation (excitation 345 nm). (B) T*/N* ratio (magenta),
scattering at 355 nm (dark cyan), and ThT signal (black dashed line, fit
according to ref 10); ThT signal of the control WT sample (red squares).
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The large increase in the T*/N* ratio (Figure 2B) and quantum
yield (SI) as well as the red-shifted T* band exhibited by AS140C-
MFC upon aggregation reflect a transfer of the probe to a
significantly less protic (H-bonding) and moderately less polar
environment. This conclusion is based on the comparison of the
properties of the aggregated protein with those reported for the furyl
chromone in aprotic solvents, particularly DMF and acetonitrile.11b
The conjugate of MFC with acetylcysteine also had a much a higher
T*/N* ratio (∼17) in DMF than in aqueous solution (SI). It follows
that the spectral changes in AS140C-MFC during thermal-induced
aggregation of AS to both I and AF reflect a diminished exposure
of the C-terminus to the aqueous solution. The discrepancies in
the temporal evolution of the MFC signals compared to those of
ThT and light scattering also suggest that at least some I lack the
aromatic-hydrophobic groove spanning the consecutive ꢀ-strands
proposed to constitute the minimal ThT binding site of amyloid
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significant ꢀ-sheet secondary structure (SI). MFC derivatives of
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