Oxidation inhibits amyloid fibril formation of transthyretin
Simin D. Maleknia
1
, Nata
`
lia Reixach
2
and Joel N. Buxbaum
2
1 School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW, Australia
2 Division of Rheumatology Research, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA, USA
Protein oxidation has been implicated in a wide range
of diseases, and ageing [1–4]. Reactive oxygen species
(ROS) contribute to processes that induce irreversible
structural damage and alter protein activity. Oxygen-
containing radicals, in particular the hydroxy radical,
react with proteins through hydrogen abstraction,
addition and elimination reactions at both the amino
acid side chains and backbone amide bonds to produce
oxidized, degraded, and cross-linked proteins [2,5,6].
The oxidized cross-linked products and protein aggre-
gates have been identified as insoluble proteins in
many diseased tissues including amyloid fibrils [7,8].
We are investigating the role of amino acid side chain
oxidation in amyloid assemblies by comparing the
kinetics of fibril formation of native and oxidized
proteins.
Interactions between amino acid side chains help to
stabilize protein structures and control folding and the
assembly of complexes [9,10]. The nature of amino
acid side chain bonds and their thermodynamic stabil-

were allowed to react over a time range of 10 min to 12 h with hydroxy
radical and other reactive oxygen species. In these timescales, up to five
oxygen atoms were incorporated into WT and V30M TTR proteins.
Oxidized proteins retained their tetrameric structures, as determined by
cross-linking experiments. Side chain modification of methionine residues
at position 13 and 30 (the latter for V30M TTR only) were dominant oxi-
dative products. Mono-oxidized and dioxidized methionine residues were
identified by radical probe mass spectometry employing a footprinting type
approach. Oxidation inhibited the initial rates and extent of fibril forma-
tion for both the WT and V30M TTR proteins. In the case of WT TTR,
oxidation inhibited fibril growth by  76%, and for the V30M TTR by
nearly 90%. These inhibiting effects of oxidation on fibril growth suggest
that domains neighboring the methionine residues are critical in stabilizing
the tetrameric and folded monomer structures.
Abbreviations
ROS, reactive oxygen species; TTR, transthyretin.
5400 FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS
residues and interactions that are critical in stabilizing
protein structures and folding.
The amyloidoses are a group of protein-misfolding
diseases that result from deposition of proteins nor-
mally soluble under physiological conditions [15–18].
These include Alzheimer’s disease, Creutzfeldt–Jakob
disease, familial amyloidotic polyneuropathy, familial
amyloidotic cardiomyopathy and senile systemic amy-
loidosis. Transthyretin (TTR) is a homotetrameric
plasma protein associated with the transport of thyrox-
ine and vitamin A [19]. Deposition of the wild-type
(WT) protein has been associated with senile systemic
amyloidosis [20], and more than 80 TTR variants have

tyrosine, tryptophan, proline, histidine, leucine and
lysine are most susceptible to reactions with ROS
[5,6,30,31]. When reactions are restricted to millisecond
timescales, limited oxidation of amino acid side chains
occurs without structural damage. This limited oxida-
tion method, termed radical probe mass spectometry
[6,30], has been utilized for probing protein structure
[32], folding [33] and interactions [34,35]. As the reac-
tion timescale increases, backbone cleavage and aggre-
gation reactions occur [6], resulting in the possibility of
structural damage [36]. The dose-dependent oxidation
method has been applied to the study of protein stabil-
ity and the onset of oxidative damage [36]. The present
study expands the utility of radical probe mass specto-
metry in investigating side chain interactions that are
critical in stabilizing protein assemblies.
Oxidized proteins for this study were prepared by
reaction with hydrogen peroxide [37] in a timescale
range of 10 min to 12 h. Oxidation of WT and
V30M TTR proteins in these timescales increased
their molecular masses by 80 Da, indicating that
up to five oxygen atoms were incorporated into the
protein structure. Electrospray mass spectometry
(ESI-MS) analysis also revealed that, after reaction
with hydrogen peroxide, these proteins were nearly all
oxidized (i.e. oxidized samples did not contain unre-
acted proteins). To verify that this level of oxidation
did not disturb the tetrameric structure of TTR, glu-
taraldehyde cross-linking reactions were performed
for WT and V30M TTR and their oxidized forms.

aggregates [24]. The results of measurements at 330
S. D. Maleknia et al. Oxidation inhibits amyloid fibril formation of TTR
FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS 5401
and 400 nm in this study were similar, and therefore
only the 330-nm data are discussed here. The kinetics
of fibril formation for the unoxidized proteins and
oxidized proteins resulting from the 12-h reaction with
hydrogen peroxide are shown in Fig. 1. Fibril growth
was followed as a function of time for up to 14 days.
These results show that both the unoxidized and oxid-
ized proteins could form fibrils. The absorbance meas-
urements (Fig. 1) show the normal pattern of an initial
exponential fibril growth over the 5-day period fol-
lowed by a slower growth period as a function of time.
As the concentration and buffers for all samples were
similar and the oxidized samples did not contain signi-
ficant amounts of unreacted protein, differences in tur-
bidity measurements reflect the effects of amino acid
side chain oxidation on fibril growth kinetics. Oxida-
tion had a dramatic affect on initial rates (slopes of
tangent lines to experimental curves up to t ¼ 24 h) of
fibril growth for both WT and V30M TTR. Larger
effects on the kinetics of fibril formation were seen for
oxidized V30M TTR compared to the unoxidized
V30M TTR than for oxidized WT TTR compared to
unoxidized WT TTR, consistent with the fact that in
V30M TTR there is one more methionine available for
oxidation than in WT TTR.
While fibril growth progressed over the 14 days, oxi-
dation inhibited the extent of fibril formation overall

Oxidation of amino acid side chains follows their
order of solvent accessibility when oxidative reactions
are performed in millisecond timescales [6,30–36]. The
reaction time influences the level of oxidation at each
reactive residue. The site of oxidation of amino acid
side chains was investigated after proteolysis by mass
spectometry sequencing. Methionine residues are
highly reactive and oxidize readily in the presence of
ROS [5,6,37]. The WT contains methionine at posi-
tions )1 (methionine resulting from the recombinant
preparation) and 13. V30M TTR contains an
additional methionine at position 30 [38]. These
methionine residues were highly oxidized to their
mono-oxidized and di-oxidized forms. The oxidation
of Met13 can be explained by an accessible surface
area of 22.8 A
˚
2
[solvent accessible surface area calcu-
lated for V30M TTR monomer (Protein Data Bank
entry1TTC) and based on the percentage of the
maximum possible exposure of the C-terminal Glu127
350 300
250
200 150 100
50 0
0.0
0.1
0.2
0.3

(oxidized) ⁄ A
330nm
(unoxidized)] x 100.
Oxidation inhibits amyloid fibril formation of TTR S. D. Maleknia et al.
5402 FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS
residue]. However, Met30 is not solvent accessible
and was completely oxidized [39].
Oxidation of the methionine residues to their mono-
oxidized and di-oxidized forms was confirmed by mass
spectometry sequencing. Figure 3 shows post-source
decay sequencing mass spectra for the di-oxidized
(after reaction with ROS) and unoxidized tryptic pep-
tides covering residues 23–35 for V30M TTR. The
protonated di-oxidized tryptic peptide is observed at
m/z 1430.5. Oxidation of the methionine residue is
verified, as C-terminus fragment ions from y
5
(MHVFR) to y
8
(NVAMHVFR) are shifted by 32u,
indicating the addition of two oxygen atoms on this
methionine residue. The y
1
to y
4
remain unchanged,
signifying that the C-terminal HVFR portion of this
peptide was not oxidized. The N-terminal fragment
ions b
3

Fig. 3. Post-source decay sequencing mass
spectra for (top) di-oxidized and (bottom)
unoxidized tryptic peptides showing the
oxidation of methionine after reaction of
V30M TTR with ROS.
S. D. Maleknia et al. Oxidation inhibits amyloid fibril formation of TTR
FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS 5403
altered tertiary contacts in a manner that stabilized the
oxidized tetramers. We speculate that oxidation may
have introduced new tertiary contacts that stabilized
the folded monomeric structure of the oxidized pro-
teins and inhibited the formation of the unfolded
monomer, which has been proposed [25,26] to be a
prerequisite for fibril growth. Together these effects
caused a delay in the onset of amyloid fibril formation.
Alternatively, the inhibition of fibril formation may
purely be the result of an increase in solubility of oxid-
ized proteins [6,41]. Limited oxidation increases the
hydrophilicity of proteins as determined by their elu-
tion times from hydrophobic columns [6,31,32]. On the
basis of liquid chromatography ⁄ ESI-MS analysis under
similar conditions, oxidized TTR proteins were eluted
 40 s faster than their unoxidized forms, indicating
an increase in their hydrophilicity.
These results show that amino acid side chain oxida-
tion can be used as a method of investigating regions of
proteins that are critical in the onset of amyloid forma-
tion. This study reveals that domains neighboring
methionine residues are critical in the formation of fibril
assemblies. These oxidation reactions are being followed

)1
; Sigma
Chemicals, St Louis, MO, USA) at a concentration of
2.7% peroxide. The oxidation reactions were performed at
pH 7.6 in a timescale range of 10 min to 12 h. The oxid-
ized proteins were then purified from the hydrogen perox-
ide reagent through extensive buffer exchange [10 mm
phosphate buffer (pH 7.6) ⁄ 100 mm KCl ⁄ 1mm EDTA]
with centriprep devices with 10-kDa filters (Millipore, Bill-
erica, MA, USA). The concentrations of all protein solu-
tions were adjusted to 10 lm with the sodium phosphate
buffer at pH 7.6 based on A
280
. The proteins were ana-
lyzed by liquid chromatography ⁄ ESI-MS to verify their
molecular masses and extent of oxidation. Proteins were
also digested with trypsin, and post-source decay sequen-
cing experiments identified the site of amino acid side
chain modification.
Kinetics of amyloid fibril formation
Chemical cross-linking was performed to check that the
tetrameric structure of proteins was preserved after the oxi-
dation reactions. Glutaraldehyde (25%) was added to pro-
tein solutions (10% v ⁄ v), and incubated for 4 min. The
reaction was quenched by the addition of NaBH
4
(7%
in 0.1 m NaOH). The samples were analyzed by
1D SDS ⁄ PAGE, and protein bands were visualized with
Coomassie blue stain.

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