From the Department of Haematology and
¶ Respiratory Medicine Unit, Department of Medicine,
University of Cambridge, Cambridge Institute for Medical Research,
Wellcome Trust/Medical Research Council Building, Hills Road,
Cambridge CB2 2XY, United Kingdom
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ABSTRACT |
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The mutation in the Z deficiency variant of
1-antitrypsin perturbs the structure of the
protein to allow a unique intermolecular linkage. These loop-sheet
polymers are retained within the endoplasmic reticulum of hepatocytes
to form inclusions that are associated with neonatal hepatitis,
juvenile cirrhosis, and hepatocellular carcinoma. The process of
polymer formation has been investigated here by intrinsic
tryptophan fluorescence, fluorescence polarization, circular dichroic
spectra and extrinsic fluorescence with
8-anilino-1-naphthalenesulfonic acid and
tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have
demonstrated that
1-antitrypsin polymerization is a
two-stage process and have allowed the calculation of rates for both of
these steps. The initial fast phase is unimolecular and likely to
represent temperature-induced protein unfolding, while the slow phase
is bimolecular and associated with loop-sheet interaction and polymer
formation. The naturally occurring Z, S, and I variants and recombinant
site-directed reactive loop and shutter domain mutants of
1-antitrypsin were used to demonstrate the close
association between protein stability and rate of
1-antitrypsin polymerization. Taken together, these data
allow us to propose a kinetic mechanism for
1-antitrypsin polymer formation that involves the
generation of an unstable intermediate, which can form polymers or
generate latent protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-Antitrypsin is a member of the serpin
protein superfamily that encompasses a wide range of serine proteinase
inhibitors involved in coagulation, inflammation, fibrinolysis, and the
complement cascade (1-3). The members of the superfamily have a common
tertiary structure based on a central
-sheet (sheet A), surrounded
by two other sheets (B and C) and a mobile, inhibitory reactive center loop (Fig. 1). The mechanism of action of
these proteins as proteinase inhibitors is most unusual in comparison
to other serine protease inhibitors, as cleavage of the
P1-P1' bond by non-target enzymes causes a
large rearrangement of the serpin with the loop incorporated into the A
-sheet and the P1 and P1' residues separated
by over 60 Å (4-6). The loop may be stably incorporated into the A
-sheet in the absence of cleavage by formation of the latent species (7-10). This is typically prepared by heating serpins for prolonged periods in the presence of stabilizing concentrations of sodium citrate
(8, 10, 11). The description of this conformational change from x-ray
crystal structures of the native (7, 8, 12-15), latent (7-9), and
cleaved (4-6, 16) forms of the serpins, combined with the fact that
the cleaved and latent forms have a much higher stability compared with
the native species (10, 17-20), has led to the suggestion that the
serpin fold is metastable (21, 22).
View larger version (49K):
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Fig. 1.
A ribbon representation of the
1-antitrypsin fold viewed from two
orientations with the reactive center loop in bold. Following
reactive loop cleavage and the formation of the latent species, the
loop is incorporated into the A
-sheet as s4A. Trp-194 in s3A,
Trp-238 in s2B, Cys-232 in s1B, and Glu-342 in s5A are shown, and
sheets A, B, and C and helix A are marked for orientation. The shutter
domain, which controls opening of the gap between s3A and s5A and
loop insertion, is shown as a dotted line.
The proximal hinge region of the reactive loop is centered on Glu-342
and labeled P, and the distal hinge is labeled D.
The figure was produced using MOLSCRIPT (47).
In 1992, we showed that severe deficiency of the Z variant of
1-antitrypsin (Glu-342
Lys) resulted from a
conformational transition and a unique linkage between the reactive
center loop of one molecule and a
-sheet of a second (23). This
process of polymer formation was temperature- and
concentration-dependent (23, 24), and the polymers that
formed had the appearance of "beads on a string" when visualized by
electron microscopy (25). Since this initial report, investigations
have been concerned with the characterization and classification of
clinically relevant mutations. These studies have shown that
polymerization can occur with a variety of deficiency mutants of
1-antitrypsin (25-27) and in variants of antithrombin
(28, 29) and C1-inhibitor (30, 31) in association with thrombosis and
angioedema, respectively. The structural mechanism by which serpin
self-assembly occurs has not yet been determined, but biophysical data
show that the polymeric form has an enhanced stability like that of the
cleaved form of
1-antitrypsin (10, 26, 32). Peptides
with homology to the reactive center loop can insert into the A
-sheet of the native molecule to block polymerization in
vitro (23, 24, 32), and these data suggest that polymerization
occurs by sequential insertion of the reactive loop from one molecule
into the A-sheet of another. X-ray crystal structures of a dimer of
antithrombin have revealed an alternative intermolecular linkage
between the reactive center loop of one molecule and the C
-sheet of
a second (7, 8, 33). The precise mechanism associated with disease remains uncertain, and although both A- and C-sheet linkages can form
in conditions that are dependent on the buffer (10, 34), the recent
structures of native
1-antitrypsin (13, 15) give support
to a reactive loop:A
-sheet interaction in vivo.
We report here the molecular dynamics of 1-antitrypsin
polymer formation. Such a study is important, as it is now becoming apparent that a deeper understanding of the mechanism of polymerization is required to allow the production of mimetics to control diseases caused by serpin misassembly.
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EXPERIMENTAL PROCEDURES |
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Purification of
1-Antitrypsin--
1-Antitrypsin was
purified from human plasma by 50% and 75% ammonium sulfate
fractionation, followed by thiol exchange and Q-Sepharose
chromatography as detailed previously (24).
1-Antitrypsin was also expressed in Escherichia
coli using the expression vector pWombAT (35) and was purified
from the crude E. coli cell extract by 8% and 28% w/v
PEG1 8000 fractionation. The
resulting pellet was resuspended in 20 mM phosphate buffer,
pH 6.8, containing 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 1% (v/v)
-mercaptoethanol and
subjected to PAE300 chromatographic separation, the protein being
eluted off the column by a 0-0.4 M NaCl gradient.
Fractions containing
1-antitrypsin were then further
purified using zinc-chelating Sepharose (equilibrated in 50 mM Tris-HCl, 150 mM NaCl, pH 8.5) and eluting
with a 0-75 mM glycine gradient. Finally, the fractions
containing
1-antitrypsin were loaded onto a
5,5'-dithiobis(2-nitrobenzoic acid)-charged glutathione-Sepharose
column (equilibrated in 100 mM Tris, 5 mM EDTA,
pH 8.0), and eluted with 15 ml of 5,5'-dithiobis(2-nitrobenzoic acid)/dithiothreitol (24).
The proteins were stored in 50 mM Tris, 50 mM KCl, pH 7.4, and their concentration quantified by measurements of UV absorbance at 280 nm with an extinction coefficient (1 mg/ml) of 0.53. Purity was confirmed by 7.5-15% (w/v) SDS- polyacrylamide gel electrophoresis (PAGE) and 7.5-15% (w/v) non-denaturing PAGE.
Preparation of Conformations of
1-Antitrypsin--
M
1-antitrypsin
polymers were prepared by heating plasma M
1-antitrypsin
(0.25 mg/ml) at 60 °C for 3 h as described previously (24) and
were confirmed by non-denaturing PAGE and a complete loss of inhibitory
activity against bovine
-chymotrypsin (10). Cleaved
1-antitrypsin was prepared by incubation with
Staphylococcus aureus V8 proteinase (24), which cleaves at
the P4-P5 bond of the reactive loop, and full cleavage was confirmed
by a 4-kDa band shift on SDS-PAGE. Latent
1-antitrypsin was prepared by heating at 67 °C in
0.7 M citrate for 12 h as detailed previously (10).
Fluorescence Measurements--
Fluorescence measurements were
made using a Perkin Elmer LS 50B spectrofluorimeter. Intrinsic
tryptophan fluorescence of 1-antitrypsin was measured in
20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, and 0.1% (w/v) PEG 8000, pH 7.4, using an
excitation wavelength of 295 nm and detecting photons emitted at
90o to the excitation beam. Wherever possible, the slits
controlling the intensity of the excitation light source were kept at
the minimum machine-permissible limit of 2.5 nm; any other values for
these slit widths were as detailed in the text. Emission slit widths
were varied between 2.5 and 15 nm, dependent on the experimental conditions in order to give an optimal emission signal. The majority of
experiments used a 0.5-ml cuvette with a path length of 1 cm on the
excitation axis and 0.2 cm on the emission axis. Throughout all
experiments the sample temperature was maintained by a heated water
jacket within the cuvette holder, the temperatures quoted within the
text being those within the cuvette.
Fluorescence Polarization-- Polarization experiments were carried out in 20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, and 0.1% w/v PEG 8000, pH 7.4, using the same system as that for the fluorescence measurements with the addition of plane polarizing filters between the light source and the sample and between the sample and the detector. Measurements were made on a single sample with five repeat measurements for each time point with the polarizers in the parallel and perpendicular positions. A value for the fluorescence polarization was calculated from two samples using Equation 1 (see Ref. 36).
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(Eq. 1) |
Production of Tetramethylrhodamine-5-iodoacetamide
(5-TMRIA)-labeled 1-Antitrypsin--
The single
cysteine of plasma
1-antitrypsin was labeled with
5-TMRIA according to the manufacturer's instructions (Molecular Probes, Inc.) This gave a labeling efficiency of 20%. Changes in the
fluorescence characteristics of the probe during
1-antitrypsin polymerization were measured in 20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, and 0.1% w/v PEG 8000, pH 7.4, by exciting at 543 nm and measuring light emitted at 567 nm. The results are the average
of eight polymerization experiments.
8-Anilino-1-naphthalenesulfonic Acid (ANSA) Binding--
A
saturated solution of ANSA was prepared in 50 mM Tris, 50 mM KCl, pH 7.4, and filtered through a 0.2-µm pore size
filter. Fifty µl of this stock solution was added to 500 µl of 20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, and 0.1% (w/v) PEG 8000, pH 7.4, in a
fluorescence cuvette. The experiment was initiated by the addition of
1-antitrypsin (typically 50 µl) to the cuvette to give
a final concentration of 0.1 mg/ml. The fluorescence changes of the
ANSA during
1-antitrypsin polymerization were then
measured by exciting with light at 370 nm and detecting fluorescence at 450 nm.
Circular Dichroism--
Circular dichroism (CD) experiments were
undertaken using a JASCO J-720 spectrapolarimeter. Samples were
prepared using 50 mM phosphate buffer (pH 7.4), and CD
spectra of samples were collected using a 0.05-cm path length quartz
cuvette at an 1-antitrypsin concentration of 0.5 mg/ml.
Changes in the secondary structure of
1-antitrypsin with
time and temperature were measured by monitoring the CD signal at 222 nm with the protein at 0.5 mg/ml in 50 mM phosphate, pH
7.4. The temperature within the cuvette was maintained by a
computer-controlled water bath connected to a water jacket integral to
the cuvette holder, and monitored by a sensor directly located in the
holder. Thermal unfolding experiments were performed using a heating
rate of 60 °C/h and measuring the change in signal at 222 nm. The
second derivative of the resulting data was then used to calculate the
inflection point of the transition and hence Tm.
Data Fitting-- The kinetic data were fitted to single or double exponential functions of the following type:
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(Eq. 2) |
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(Eq. 3) |
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RESULTS |
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Intrinsic Tryptophan Fluorescence--
The polymerization of
1-antitrypsin is both concentration- and
temperature-dependent (24). Plasma
1-antitrypsin was incubated over a range of temperatures
and concentrations to obtain the optimum conditions for the assessment
of rates of polymer formation by intrinsic tryptophan fluorescence. At
temperatures over 50 °C, the rates were too fast to dissect out the
different phases of polymerization, and at lower temperatures, the rate
was too slow to be assessed during a 24-h incubation. At 45 °C and
0.1 mg/ml, there was an increase in intrinsic tryptophan fluorescence (Fig. 2a) and a 2.5-nm blue
shift, which was 50% complete by 36,000 s (10 h), 80% complete by
80,000 s (22 h), and 95% complete after 151,200 s (42 h). Mathematical
analysis of the change in the fluorescence intensity with time revealed
a fast and slow exponential phase. Examination of the protein at
various time points throughout polymerization by non-denaturing PAGE
showed no high molecular mass species in the first 7200 s (2 h;
Fig. 2b) and then a rate of decrease in intensity of the
native band, which agreed well with the slower rate calculated from
intrinsic tryptophan fluorescence (Fig. 2c). In order to
demonstrate the specificity of these signals, the experiment was
repeated with
1-antitrypsin cleaved at the reactive center loop. This form is unable to undergo polymerization (24) and
showed no change in tryptophan fluorescence when incubated under the
same conditions. The phenomena reported by the tryptophan fluorophores
during the fast and slow phase of polymerization were assessed with a
variety of other spectroscopic methods (Table I).
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Fluorescence Polarization--
Measurement of the polarization of
the fluorescence from tryptophans within the protein showed a decrease
in fluorescence polarization with time (Fig.
3a). This indicates an
increase in the speed of motion of the tryptophans and may be explained
by either an increase in the rate of motion of this moiety within the
protein scaffold or a decrease in tumbling time of the protein overall
(36). The rate of decrease in fluorescence polarization over 6000 s was similar to the rate of the fast phase observed with intrinsic
tryptophan fluorescence (Table I). The slow phase was not resolved with
this technique.
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Extrinsic Fluorescence--
The changes in properties of
1-antitrypsin during polymer formation were assessed
following labeling of cysteine 232 (Fig. 1) with an extrinsic rhodamine
probe, 5-TMRIA. The fluorophore reported an increase in fluorescence
and a 2.0-nm blue shift with a rate constant similar to that of the
slow phase detailed previously (Fig. 3b). The fast phase
could not be resolved with this technique. These data indicate that the
fluorophore at position 232 has become more protected from solvent as
the protein polymerizes.
The dye ANSA was also used to probe the conformational changes of
1-antitrypsin during polymerization, as its fluorescence is sensitive to changes in protein surface hydrophobicity (37). Measurements of ANSA fluorescence during polymerization show an initial
small fast fluorescence enhancement (Fig. 3c), followed by a
larger decrease in fluorescence which occurs at a rate close to that of
the slow phase. These data show that in the early stages of the
experiment
1-antitrypsin transiently exposes hydrophobic domains before the slow reburial of these exposed residues.
Circular Dichroism--
Changes in the far-UV CD spectra were
observed between the five-stranded native and the six-stranded reactive
loop cleaved and latent forms of 1-antitrypsin (Fig.
4a). An increase in the magnitude of the signal at 222 nm was observed as
1-antitrypsin formed polymers when incubated at 0.1 mg/ml and 45 °C (Fig. 4b). These data were the mean of
five experiments and allowed the calculation of a fast phase (Table I),
which closely matched that observed by intrinsic fluorescence. Spectra
were also taken at the beginning of the experiment and after 24 h
when the polymerization was 80% complete as assessed by change in
fluorescence signal (Fig. 4a). This shows a decrease in
intensity at 222 nm (the region associated with
-helix content) of
only 0.25 × 10
3 millidegrees cm2
dmol
1, which is compatible with
1-antitrypsin polymerization. Polymers were also
prepared by heating
1-antitrypsin at 0.25 mg/ml at 50 °C for 12 h in 50 mM Tris, 50 mM
KCl, pH 7.4 (Fig. 4c). The far-UV CD profile of these
polymers was significantly more negative than that of native
1-antitrypsin and
1-antitrypsin polymers formed at 45 °C (Fig. 4a).
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The Concentration Dependence of the Fast and Slow Phase of
Polymerization--
Previous studies have shown that the overall rate
of 1-antitrypsin polymer formation was dependent on
protein concentration (24). The fast phase of the fluorescence signal
during polymerization was unaffected by protein concentration,
confirming that the process was unimolecular (data not shown). The slow
rate did show dependence on concentration, which became non-linear at
high protein concentrations (Fig. 5).
Incubation of
1-antitrypsin at 0.1 mg/ml and 45 °C with stabilizing sodium citrate, which favors the formation of the
latent protein, reduced the rate of polymerization by approximately 15-fold (Fig. 5).
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pH Dependence of Polymerization--
The results obtained during
these studies suggest that increasing the conformational flexibility of
1-antitrypsin plays an important role in increasing the
proteins' propensity to polymerize. This was further assessed by
changing the solution pH to destabilize the protein and assessing the
rate of polymerization with intrinsic fluorescence. The rate of
polymerization was measured at pH 5, 6, 7, 8, and 9 using buffers with
protonation states that were insensitive to temperature changes. Rates
of polymerization were considerably increased at high and low pH (Fig.
6a), and this was associated
with reduced melting temperatures as assessed by CD spectra at 222 nm
(Fig. 6b).
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Effect of Point Mutations on Rates of Polymerization--
Our
interest in the polymerization of 1-antitrypsin results
from its association with plasma deficiency, cirrhosis, and emphysema (38). Thus detailed studies of the effect of medically relevant mutations on the protein's behavior are important. In this study, we
have taken advantage of the availability of such mutants and have
studied their propensity for polymerization using the methods detailed
above. Six
1-antitrypsin variants were used in this study, which represent both naturally occurring variants purified directly from blood plasma (Z, S, I, and M
1-antitrypsin) and mutations introduced into a
recombinant framework (wild type, Phe-51
Leu, Glu-354
Gln, and
Glu-354
Ser
1-antitrypsin) and expressed in E. coli. Analysis of these variants showed that an increased rate of
polymer formation by the protein correlated with a reduced melting
temperature (Table II).
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DISCUSSION |
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The primary objective of this study was a detailed analysis of the
process of 1-antitrypsin polymer formation. The data
from tryptophan fluorescence and the extrinsic probe ANSA showed that there were two processes with 10-fold different rates during polymer formation. The faster of the two processes was independent of protein
concentration and was observed as the sole process during the
measurement of fluorescence polarization and circular dichroism. This
is consistent with a conformational change occurring within the protein
induced by a change in temperature. This process did not alter the
fluorescent characteristics of the rhodamine probe bound to Cys-232 in
the C-sheet, implying that the conformational change occurs elsewhere
in the protein. The change in tryptophan fluorescence must result from
the perturbation of tryptophan residues at either 194 or 238 (Fig. 1).
Trp-238 is on the surface of
1-antitrypsin, 6.5 Å from
Cys-232, with no stable hydrogen bonds visible in the native structure
(13, 15). It seems improbable that a conformational change within the
protein could increase the fluorescence of Trp-238, and indeed
site-directed mutagenesis has shown that this residue makes no
contribution to tryptophan fluorescence on refolding of
1-antitrypsin (39). Thus, increasing temperature
perturbs the environment of Trp-194 in
1-antitrypsin,
reflecting disorganization of the strands at the top of the A
-sheet.
The slower of the two processes was also observed as changes in tryptophan and ANSA fluorescence and the fluorescence of rhodamine linked to Cys-232. This process is dependent on protein concentration and was associated with the formation of high molecular mass loop-sheet polymers on non-denaturing PAGE. The fact that the fluorescence of a moiety attached to the C-sheet is affected during polymer formation is consistent with the proximity of the residue to the reactive loop, which provides the "linker" segment in models of loop-sheet polymers (13, 40). Although the slow phase of polymer formation was dependent on protein concentration, at high concentrations the process was saturated (Fig. 5). This suggests that, at high concentrations, a second process, such as nonspecific aggregation competes with polymer formation. On the basis of these results, we propose the following kinetic mechanism for the polymerization of a serpin.
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If polymer formation is a two-step process, then we would expect the
rate of conversion from M to P (k2) to be
dependent on the concentration of M* and hence the rate of M M*
(k1). Our data show that the difference in rates
between the two processes is on the order of 10-fold (Table I). Thus,
following the initiation of the reaction, there should be a short lag
in the production of P as the concentration of M* rises. Two of our
experiments measure only the production of P: non-denaturing PAGE and
rhodamine fluorescence. Rhodamine fluorescence indeed shows a small lag in the increase in signal (Fig. 3b, inset) in the
early stages of the experiment, which would agree well with a build-up
of M*. As further proof, comparisons of simulations of the production of P with time (using numerical solutions to differential equations describing the above mechanism), with the data from rhodamine fluorescence show good agreement. Parameters extracted from these simulations also give a value for k1 (5.2 × 10
4 s
1) that is very similar to those
shown in Table I. Further to this, non-denaturing gels shows no higher
molecular weight species forming in the first 7200 s, again
suggesting a lag in the formation of polymers. Taken together, these
data support the proposal that polymerization is a sequential two-step
process: an initial fast conformational change within the protein
producing a polymergenic intermediate, which then undergoes loop-sheet linkage.
Incubation of 1-antitrypsin with sodium citrate favors
the formation of the latent species (10, 11, 34). In order to investigate this,
1-antitrypsin was incubated with
sodium citrate. The concentration of citrate (0.7 M)
routinely used to produce latent
1-antitrypsin (17)
disrupted fluorescence measurements due to solution turbidity. A lower
concentration of citrate was therefore used (0.5 M), which
produced insufficient latent
1-antitrypsin to be
visualized by non-denaturing PAGE. Despite this, the citrate reduced
the rate of polymerization by an average of 15-fold (Fig. 5). This
suggests that the action of citrate (even when present at a suboptimal
concentration) was to reduce the value of k2 and favor the formation of latent protein.
This kinetic scheme also allows the interpretation of the effects of
destabilization of the protein by extremes of pH and mutations on the
rate of conformational change (Fig. 6 and Table II). Any factor that
decreases protein stability (as assessed by a reduced melting
temperature) will result in an increase in the equilibrium
concentration of M* and so will increase the formation of
both polymers and the latent conformation. Naturally occurring
deficiency mutants of the serpins cluster in the shutter domain
(Siiyama and Mmalton 1-antitrypsin) and the proximal (Z
1-antitrypsin) and distal hinge regions (38). These have
been predicted to open the A
-sheet of the protein, thereby
increasing M* and favoring the formation of polymeric and latent
protein at lower activation temperatures (21, 38). Mutants that
increase the stability of the serpins (such as Phe-51
Leu
1-antitrypsin) will reduce M* and so reduce conversion to the latent or polymerized conformation (22, 41). Thus, it is
possible to explain the observation that mutant serpins with reduced
thermal stabilities more readily undergo polymerization (Table II).
Circular dichroism may be used to assess conformational transitions of
proteins and major differences are apparent in the far UV spectra
between the native (five-stranded) and cleaved (six-stranded) forms of
the inhibitory serpins (18). This technique was therefore used to
assess a second six-stranded species, latent 1-antitrypsin, with the anticipation that it would be
similar to the cleaved form (Fig. 4a). Surprisingly, the CD
profile of latent
1-antitrypsin was very different from
cleaved
1-antitrypsin, which suggests that the reactive
loop may not be fully inserted into the A
-sheet in the latent
species or that the position of s1C is important in the far-UV CD
profile, as it will be fixed in the C-sheet in the cleaved structure
(4) but must be displaced in latent
1-antitrypsin (8).
The change in CD signal at 222 nm was also used to assess the
conformational change that was associated with polymerization and gave
a fast rate that was very similar to that obtained by intrinsic
fluorescence (Fig. 4b and Table I). The far-UV CD profile of
1-antitrypsin polymerized at 45 °C for 24 h was
markedly different from that obtained after polymerizing
1-antitrypsin at 50 °C and 0.25 mg/ml for 12 h
(Fig. 4a). The polymers formed at 50 °C are longer (10),
and the tendency to aggregate makes it difficult to accurately
determine protein concentration and thereby the position of the CD
profile in relation to native
1-antitrypsin.
The phenomena of proteins self-assembling to form a polymeric structure
may be mechanistically classified into two subtypes (42). The first
type represents those most commonly encountered in which self-assembly
is mediated by surface interactions between folded proteins. This sort
of interaction causes sickle cell hemoglobin to polymerize and proteins
to crystallize. The second type of self-assembly has been studied with
great interest over the past few years, as it is central to the
mechanisms of the transmissible spongiform encephalopathies and
Alzheimer's disease. The self-assembly event of
1-antitrypsin fits well with this form of
oligomerization (43), as
1-antitrypsin polymerization,
like the amyloid protein transthyretin (44-46), is enhanced by
destabilizing the structure of the protein with extremes of pH and
point mutations. It also seems reasonable to think of native
1-antitrypsin as a folding intermediate (21, 22), albeit
a very stable one with the end product of the folding pathway being the
six-stranded latent form of the protein.
In summary, we present here a detailed analysis of the processes
underlying the polymerization of 1-antitrypsin, linking a variety of spectroscopic signals to changes that occur within the
protein. We have also investigated the effect that mutations which
interfere with these processes have on polymerization, and have
reinforced the link between serpin polymerization and amyloid-like self-assembly.
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ACKNOWLEDGEMENTS |
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We are grateful to James A. Huntington and Robin W. Carrell for critical appraisal of the manuscript.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust, Medical Research Council (United Kingdom), the Cystic Fibrosis Trust, and Papworth National Health Service Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 44-1223-336829; Fax: 44-1223-336827; E-mail: td214{at}cam.ac.uk.
A Medical Research Council Training Fellow.
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ABBREVIATIONS |
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The abbreviations used are: PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; 5-TMRIA, tetramethylrhodamine-5-iodoacetamide; ANSA, 8-anilino-1-naphthalenesulfonic acid.
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REFERENCES |
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