Subunit Exchange Demonstrates a Differential Chaperone Activity
of Calf
-Crystallin toward
LOW- and Individual
-Crystallins*
Tatiana
Putilina,
Fériel
Skouri-Panet,
Karine
Prat,
Nicolette H.
Lubsen
, and
Annette
Tardieu§
From the Laboratoire de Minéralogie-Cristallographie, CNRS
and P6-P7 Universities, Case 115, 4 Place Jussieu, F75252 Paris
Cedex 05, France and the
Department of Biochemistry 161, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The
Netherlands
Received for publication, August 9, 2002, and in revised form, January 30, 2003
 |
ABSTRACT |
The chaperone activity of native
-crystallins
toward
LOW- and various
-crystallins at the
onset of their denaturation, 60 and 66 °C, respectively, was studied
at high and low crystallin concentrations using small angle x-ray
scattering (SAXS) and fluorescence energy transfer (FRET). The
crystallins were from calf lenses except for one recombinant human
S. SAXS data demonstrated an irreversible doubling in molecular
weight and a corresponding increase in size of
-crystallins at
temperatures above 60 °C. Further increase is observed at 66 °C.
More subtle conformational changes accompanied the increase in size as
shown by changes in environments around tryptophan and cysteine
residues. These
-crystallin temperature-induced modifications were
found necessary to allow for the association with
LOW-
and
-crystallins to occur. FRET experiments using IAEDANS
(iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF
(iodoacetamidofluorescein)-labeled subunits showed that the
heat-modified
-crystallins retained their ability to exchange
subunits and that, at 37 °C, the rate of exchange was increased
depending upon the temperature of incubation, 60 or 66 °C.
Association with
LOW- (60 °C) or various
-crystallins (66 °C) resulted at 37 °C in decreased subunit
exchange in proportion to bound ligands. Therefore,
LOW-
and
-crystallins were compared for their capacity to associate with
-crystallins and inhibit subunit exchange. Quite unexpectedly for a
highly conserved protein family, differences were observed between the
individual
-crystallin family members. The strongest effect was
observed for
S, followed by h
Srec,
E,
A-F,
D,
B.
Moreover, fluorescence properties of
-crystallins in the presence of
bound
LOW-and
-crystallins indicated that the
formation of
LOW/
- or
/
-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of
-crystallins toward the
other lens crystallins demonstrate the dynamic character of the
heat-activated
-crystallin structure.
 |
INTRODUCTION |
-Crystallins and small heat shock proteins have in
common a conserved C-terminal domain of about 115 residues, the
-crystallin domain, the structure of which is organized around an
eight-stranded
-sandwich (1, 2). The small heat shock proteins
usually associate into high molecular weight monodisperse or
polydisperse oligomers, able to protect against stress through the
binding of a variety of partially unfolded substrates.
-Crystallin
was demonstrated by Horwitz in 1992 (3) to also exhibit chaperone properties in vitro. It is able to bind
- and
-crystallins at the onset of their thermal denaturation, thus
preventing further denaturation and aggregation, yet not refolding.
Following this pioneering work, the chaperone-like activity of
-crystallin has been the subject of numerous studies and functional
models have been suggested (4-30). Essentially, hydrophobic patches on
the surface, either present in the native state or revealed after structural modifications, would associate with a variety of partially denatured substrates from insulin or
-lactalbumin to the other lens
proteins, in a substrate-dependent manner. Moreover, the dynamic character of the
-crystallin quaternary structure with the
occurrence of temperature dependent subunit exchange was demonstrated and analyzed (31, 32). Fluorescence resonance energy transfer (FRET)1 was found
particularly useful to demonstrate that subunits, from recombinant
A- or
B-crystallin, can also reversibly exchange between
oligomers (31, 33-36). Subunit exchange was also detected for other
small heat shock proteins such as Hsp27, Hsp16.9, and Hsp16.5 (2, 37).
The studies also demonstrated that subunit exchange might be partially
inhibited in the presence of the bound substrates.
In the physiological context of the lens, such properties of
-crystallins would be advantageous in protecting against cataract by
delaying the formation of light-scattering aggregates, which affect
lens transparency. Yet, only a few chaperone studies with
- and
-crystallins as substrates have been performed (4, 5, 38-41)
without any correlation with subunit exchange and without any attempt
to analyze possible differences between different members of the
B-
-crystallin family. It is therefore the aim of this paper to
further investigate these points.
This study was performed with native calf crystallins and one human
recombinant
S (h
Srec). Some of our preliminary experiments and
recently published data (42-44) indicated that, at the high temperatures necessary for the chaperone activity of
-crystallin toward other lens proteins, an important conformational change in
-crystallin structure takes place. Therefore, the
temperature-induced structural changes in
-crystallin were analyzed
in relation to its subunit exchange properties. Possible differences in
-crystallin behavior toward different lens proteins as substrates
were evaluated by FRET using IAEDANS- and IAF-labeled subunits. The
rates of
-crystallin subunit exchange were compared depending on
bound 
-crystallins.
 |
EXPERIMENTAL PROCEDURES |
Materials--
5-(Iodoacetamido)fluorescein (5-IAF),
5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic
acid (1,5-IAEDANS), tris-(2-cyanoethyl)phosphine, and
tris-(2-carboxyethyl)phosphine, hydrochloride were purchased from
Molecular Probes (Eugene, OR). All other chemicals were of analytical grade from Sigma.
Purification of Calf Lens Crystallins--
The
-,
-, and
-crystallins were prepared from young calf lenses as described
before (45, 46). Briefly, the cortex was separated from the nucleus by
a gentle stirring of the lens in 150 mM phosphate buffer,
pH 6.8 (22 mM Na2HPO4, 28 mM KH2PO4, 200 mM KCl,
1.3 mM EDTA, 3 mM NaN3, 3 mM dithiothreitol) for 15-30 min at 6 °C. The
-,
LOW-, and cortical
- (
C) crystallins were prepared from the clear supernatant fraction of the cortical extracts by gel filtration, using a Amersham Biosciences FPLC system and a Superdex S-200 PG column. The nuclear
-crystallins (
N) were purified in the same way from the nuclear extracts. The individual
-crystallins were further purified by cation exchange chromatography on a Mono S HR 10/10 column. Crystallin concentrations were calculated using molar extinction coefficient of 0.85 cm2
mg
1 for
-crystallin, 2.3 cm2
mg
1 for
LOW-, 1.85 cm2
mg
1 for
S-, 2.05 cm2 mg
1 for
E-, 2.1 cm2 mg
1 for
D-,
2.02 cm2 mg
1 for
B-, and 2.0 cm2 mg
1 for
A-F-crystallin (that elute together).
Expression and Purification of Human
S-crystallin--
The
recombinant human
S (h
Srec) was prepared as described
previously (47). In short, the coding sequence was amplified from a
human lens cDNA library and cloned NdeI/BamHI
in the pET3a vector. The expression construct was introduced into the
BL21(DE3) pLysS bacterial strain, and protein expression was induced by addition of isopropyl-
-D-thiogalactopyranoside.
The protein was extracted from cells using standard procedure and
purified by gel filtration using a Sephacryl S200 HR column. The purity
of the recombinant protein was checked by SDS-PAGE electrophoresis and
mass spectrometry.
Chaperone Activity Assays--
The chaperone-like activity of
-crystallin was monitored by measuring the absorbance at 360 nm as a
function of time. Measurements were made every minute during half an
hour with a UV/VIS LambdaBio spectrophotometer. The concentration of
-crystallin remained at 20 mg/ml and 
-crystallins were added
at different w/w ratios. The temperature inside the cuvette was
thermostated with a Ministat water bath and was verified prior to data
collection using a Checktemp 1 microthermocouple thermometer. The
soluble fractions were examined by gel filtration through a Sephacryl
S200 HR column, using a Amersham Biosciences FPLC system.
Gel Electrophoresis and Western Blot Analysis--
Samples were
prepared by mixing proteins v/v with the buffer A (125 mM
Tris, pH 6.8, 20% glycerol, 4% SDS, 1%
-mercaptoethanol) and
boiling (48). SDS-PAGE fractionation was performed on 15% polyacrylamide gels. Proteins were blotted to polyvinylidene difluoride membrane (Bio-Rad) by capillarity in the buffer B (10 mM
Tris pH8.2, 2 mM EDTA, 50 mM NaCl, 2 mg/l
dithiothreitol) and immunoreactions were carried out as
described in the immunoblotting kit manual (Anti-rabbit IgG, Alkaline
Phosphatase; Calbiochem) with anti-
-crystallin polyclonal antibodies
at dilution 1:500.
Small Angle X-ray Scattering (SAXS)--
The intensity scattered
by one particle as a function of the scattering vector s
(where s = 2sin
/
and 2
is the scattering angle), usually called the particle form factor, is the Fourier transform of the spherically averaged autocorrelation function of the
electron density contrast associated with the particle. When the
solution is ideal, i.e. in the absence of interactions, the
total scattered intensity, I(s), is the sum of
the scattering of the individual particles, and the scattering near the
origin can be written (49): I(s) = exp[
4/3
2R
s2];
therefore, "Guinier plots," i.e. plots of log
I(s) versus s2,
provide us with the extrapolated intensity at the origin,
I(0), which is proportional to the molecular weight and with
the radius of gyration, Rg, which is a function of
the particle size. The experiments were carried out using the small
angle instrument D24 using the synchrotron radiation emitted by the
storage ring DCI at the Laboratory for Synchrotron Radiation,
L.U.R.E. (Orsay, France). Data were collected using a linear
position-sensitive detector with a delay line readout. The
s-increment per channel was 0.000146 Å
1, and
the average recorded s range was 0.2 × 10
2 < s < 4 × 10
2
Å
1. The wavelength of the x-rays was 1.488 Å (K-edge of
nickel). The solution experiments were performed with a
specially designed quartz cell operated under vacuum (50) that could be
filled and rinsed in situ. The intensity curves,
I(c,s), were subtracted for background
and, within each series, scaled on the same relative value with a
normalization for concentration. No scaling was done between series.
Labeling of Crystallins with Fluorescent Probes--
The
labeling reaction was carried out at a final protein concentration of
about 1 mg/ml in 150 mM phosphate buffer, 10 mM EDTA, pH 6.8, following the same protocol for both probes. Prior to
labeling, a 10-fold molar excess of each reducing agents
tris-(2-carboxyethyl) phosphine hydrochloride and
tris-(2-carboxyethyl)phosphine were added to assure the reduction of
disulfide bonds. Each probe was solubilized in methanol, and a small
aliquot was added for a final ratio of 20 mol of probe/mol of protein.
The reaction then proceeded for 4 h at 37 °C, followed by
overnight incubation at 4 °C. The labeling was terminated by the
addition of a 10-fold molar excess of mercaptoethanol. A vacuum
concentrator unit with 100,000 and 10,000 cut-off membranes were used
to remove any unconjugated probe until no fluorescence could be
detected in the eluate for
- and
-crystallin, respectively. The
molar ratio of attached probe was calculated from the absorbance
spectrum of the labeled protein and that of a free probe. Typically,
all Cys residues in
-crystallin could be labeled with either probe.
For
-crystallin two residues per molecule could be modified with
IAEDANS.
Steady State Fluorescence--
Fluorescence measurements were
made with SPF-25 spectrofluorometer using quartz cuvettes with a 10-mm
path length. Excitation and emission slits were set to 5-nm bandwidth.
Data were corrected for the contribution of solvents. For temperature
dependence studies, the cells were thermostated with a Ministat water
bath. The temperature was verified within each cuvette prior to data
collection using a Checktemp 1 microthermocouple thermometer. All
samples were in phosphate buffer, pH 6.8, unless specified.
Microcuvettes with stopper were used for incubation at high
temperatures to avoid evaporation.
Subunit Exchange--
Experiments on subunit exchange of
-crystallin were based on the protocol of Bova et al.
(31) with the following modifications. Two populations of
-crystallin labeled with 1,5-IAEDANS (donor) or 5-IAF (acceptor)
were heated at 60 or 66 °C for 30 min, either alone or in the
presence of other crystallins, and all samples were returned to room
temperature afterward. The rate of subunit exchange was evaluated from
energy transfer data. Donor and acceptor labeled
-crystallins were
mixed at 37 °C, and the degree of energy transfer was estimated over
time by following the decrease in the intensity of the donor emission.
Wavelengths of excitation and emission were 335 and 460 nm. The rate
was calculated from the equation
F(t)/F(0) = A + Be
kt, where k is defined as
the transfer rate constant, and
F(t)/F(0) corresponds to the emission
intensity ratio at time = t and 0, respectively (34).
The efficiency of energy transfer (E) at different time
points was calculated from the relationship to the intensity of the
donor fluorescence in the absence (Fa) and presence (Fd) of the acceptor: E = 1
(Fd/Fa).
 |
RESULTS |
Labeling of
-Crystallin with Fluorescent Probes--
Two
populations of
-crystallins were labeled covalently at the cysteine
residues with either IAEDANS or IAF. IAEDANS and IAF are fluorescent
probes that have been well characterized over the past years (51, 52).
They are of relatively small size and are not known to induce
conformational changes upon binding to proteins. Molar extinction
coefficients of 5700 (IAEDANS), 75,000 (IAF), and 0.8 (
-crystallin,
1 mg/ml) were used to estimate the molar ratio of the covalently
attached probe. Calf
-crystallin oligomers consist of
A (2/3) and
B subunits (1/3).
B has no cysteine residues but all cysteine
residues at a single site at Cys-131 (beginning of strand
8
according to (1)) on
A subunit could be labeled. The integrity of
labeled
-crystallin in our experiments was confirmed by unchanged
tryptophan fluorescence and accessibility to acrylamide (results not
shown). Labeling of
S (molar extinction coefficient, 1.85) with
IAEDANS resulted in the addition of two fluorophore molecules per one
S, i.e. some of the five Cys residues could be modified.
IAEDANS are preferentially reactive with cysteine residues in
hydrophobic environment, and the labeling is in agreement with common
structural features of
-crystallin.
-Crystallin Quaternary Structure at High Temperature as Studied
by SAXS--
The method that we have been using for a long time to
purify
-crystallin from cortical extracts of calf lenses
reproducibly provided us with oligomers that we characterized by SAXS
to be made up of about 40-45 subunits, with a radius of gyration of about 62 Å and an external diameter of 170Å (45, 53). These values
are in agreement with the molecular weight values usually reported in
the literature and with recent electron microscopy (EM) observations
(43). Yet,
-crystallin quaternary structure is known for
a long time to be sensitive to environmental conditions (53). Moreover,
our preliminary investigations and recently published studies (42, 43)
have shown that, at the high temperatures required for the chaperone
effect of
-crystallin toward other lens proteins to take place
(60 °C for the
- and 66 °C for the
-crystallin), the
-crystallin quaternary structure undergoes a major, irreversible,
conformational change. We therefore used SAXS to further investigate
the point. Purified fractions of
-crystallin were incubated at 60 or
66 °C for various periods of time, and at different protein
concentrations, from 5 to 20 mg/ml. The samples were then cooled either
to room temperature or to 4 °C, controlled by gel filtration, and
analyzed by SAXS. Typical results are shown in Fig.
1. The gel filtration profile in Fig.
1a obtained after 1 h of incubation at 60 °C
indicates that all the native
-crystallins have been transformed
into higher molecular weight particles, since they elute in the void
volume. SAXS experiments performed at 60 °C (Fig. 1b)
provided us with quantitative values. The conformational change is
achieved within about 10 min, and no reversibility whatsoever is
observed upon return to room temperature (not shown). The 60 °C
molecular weight is about twice the native one. The shape of the
scattering curve also indicates that the
-crystallin size has
increased. The increase in both size and molecular weight is even more
pronounced after incubation at 66 °C (Fig. 1b). The radii
of gyration of native and of 60 and 66 °C incubated samples were,
respectively, 61.4, 82.3, and 98 Å. These observations are in
agreement with the EM data that show much bigger particles after
incubation at high temperatures (43). The shape of the SAXS intensity
curves at low angles, however, is not consistent with the presence in
our experimental conditions of chain-like structures, as seen by EM
(43). Experiments performed with different incubation times (not shown)
indicate that the high temperature quaternary structure remains stable
for at least 1 h. Yet, the molecular weight and size may continue
to grow, albeit slowly, with prolonged incubation time, e.g.
overnight incubation at 66 °C usually leads to whitish
precipitates.

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Fig. 1.
The chaperone-like activity of
-crystallin at elevated protein
concentrations. a, gel filtration profile of
-crystallin at room temperature before ( ) and after ( )
incubation for 1 h at 60 °C; b, x-ray scattering
intensities of -crystallin (10 mg/ml) at room temperature
( ), 60 °C ( ), 66 °C (t); c, gel filtration
profile of and LOW mixtures (2:1, w:w) at room
temperature before ( ) and after ( ) incubation at 60 °C for 60 min; d, x-ray scattering intensities for -crystallin (10 mg/ml) in the absence ( , room temperature; , 60 °C) and
presence ( , room temperature; , 60 °C) of LOW
(2:1, w:w); e, gel filtration profile of - and
C-crystallin mixtures (4:1, w:w) at room temperature before ( )
and after ( ) incubation at 66 °C for 60 min; f, x-ray
scattering intensities of -crystallin ( , room temperature; ,
66 °C), - and C, 4:1, w:w ( , room temperature; ,
66 °C), - and N, 4:1, w:w ( , room temperature; ,
66 °C); g, temperature effect on the aggregation state of
different crystallins followed by the absorbance (360 nm) at 60 °C
for -crystallin ( ), LOW ( ), N ( ), C
( ), and at 66 °C for - ( ) and N ( ); h,
chaperone activity assay for different mixtures of lens crystallins.
The absorbance at 360 nm was recorded during the incubation of with
LOW (2:1, w:w, 60 °C, ), with N(4:1, w:w,
66 °C, ), with C (4:1, w:w, 66 °C, ); i, SDS
gel electrophoresis: lane 1, -crystallin; lane
2, C-crystallin; lane 3, - and C-crystallin
mixture at room temperature; lane 4, and C high
molecular weight peak (Fig. 1e). k, Western blot
of the same samples, using -crystallin antibodies; positive staining
in high molecular fraction (lane 4) confirms the association
of - and -crystallins.
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Temperature Effect on Lens Crystallins as Observed by
Fluorescence--
The effect of temperature on lens crystallins was
also evaluated by steady state fluorescence techniques. The decrease in the quantum yield of tryptophan fluorescence with increase in temperature was observed for all crystallins studied (Fig.
2). Yet, the overall decrease did not
reach the maximum possible for free tryptophan in solution (Fig.
2g). Together with the fact that the maxima of Trp
fluorescence were only slightly red shifted, this indicates that all
crystallins essentially retain their tertiary structure at temperatures
up to 66 °C. The result is in agreement with infrared spectroscopy
studies, which show that secondary structures are essentially preserved
(39). On return to ambient temperatures, however, the changes were not
totally reversible reflecting different stability of the lens proteins
(Fig. 2).
-Crystallin displayed an increase in quantum yield, with
the maximum of emission returning to 333 nm (Fig. 2a).
LOW-Crystallin displayed a larger increase in quantum
yield, and the red shift in maximum of emission was not reversible
(Fig. 2b), whereas
-crystallin exhibited good
reversibility in Trp emission (Fig. 2, c and d). All experiments using fluorescence techniques were conducted at concentrations below 0.05 mg/ml, and in contrast to the high protein concentration observations, no insoluble precipitated
LOW- or
-crystallin could be detected after
incubation at 60 or 66 °C for up to 30 min. However the
appearance of a Rayleigh peak at 55 °C for
LOW-crystallin and at 66 °C for
-crystallin was an indication of light scattering by larger, soluble, protein aggregates (Fig. 2). Such a difference in the onset of the Rayleigh peak reflects
differential responses to heat stress among
- and
LOW-crystallins.

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Fig. 2.
Spectral properties of lens proteins at
different temperatures. Fluorescence spectra were recorded using
tightly closed cuvettes after incubation for 30 min at 37 °C ( ),
60 °C ( ), 66 °C (o), and 37 °C on return from high
temperature ( ). Concentrations of proteins were adjusted to ensure
the absorbance below 0.05 at the wavelength of excitation.
Monochromator bandwidth of 5 nm was used in all experiments.
a, -crystallin; b,
LOW-crystallin; c, S-crystallin;
d, B-crystallin; e, -IAF; f,
-IAEDANS; g, L-tryptophan as control in
phosphate buffer; h, normalized emission spectrum of
-IAEDANS, ex = 335 nm ( ), and the excitation
spectrum of -IAF, em = 550 nm ( ). The overlap
between spectra illustrates the feasability of FRET between labeled
molecules. Emission spectra of protein tryptophan fluorescence were
collected using ex = 295 nm; emission spectra of
-IAEDANS and -IAF were collected using ex = 335 nm
and ex = 460 nm.
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A Rayleigh peak is also observed with
-crystallin at the onset of
the conformational change at 60 °C. Moreover, changes in extrinsic
fluorescence of labeled
-crystallin cysteine residues are
illustrated by emission spectra of
-IAEDANS and
-IAF at 37 °C,
before and after incubation at 66 °C for 30 min (Fig. 2, e and f). As can be seen from Fig. 2f,
the incubation of
-crystallin leads to a decrease in hydrophobicity
around cysteine residues that is only partially reversible after the
return to ambient temperature. Such an increase is consistent with an
increase in molecular size. Because of its small Stokes shift (spectral
distance between absorption and emission maxima), the fluorescence of
fluorescein compounds is known to be quenched in proteins by
homotransfer (51). The overall increase in the average distance
distribution between labeled cysteines would release the quench,
resulting in increased fluorescence yield. Overall, the data on
-crystallin are consistent with minor changes in the secondary and
tertiary structure of the subunits and with the size increase that has been demonstrated by SAXS.
The Chaperone Activity of
-Crystallins Is Associated with the
Formation of Heterocomplexes--
The chaperone-like activity of the
-crystallin versus
LOW- and
-crystallin, i.e. the ability to prevent temperature
induced precipitation of the
LOW/
-crystallin, was
studied at 60 and 66 °C, respectively, by measuring the absorbance
as a function of time at 360 nm (3, 4), with protein concentrations
varying from about 2 mg/ml up to 20 mg/ml. Most of the assays reported in the literature have been performed at protein concentrations of
about or below 0.5 mg/ml. The use of much higher protein concentrations would be more relevant to the in vivo conditions. It allowed
us to confirm the chaperone activity of
-crystallin at elevated protein concentrations as well (Fig. 1h). Gel filtration
analysis and SAXS studies were also performed at the same high protein concentrations facilitating the comparison of all data on chaperone activity. In this study the total
-crystallin extracts
N (from nucleus) or
C (from cortex) were used. The
LOW-,
N-, and
C-crystallin alone were found to precipitate rapidly, in
5-15 min (Fig. 1g). We indeed found, as already reported in
the literature, that
-crystallin easily prevents
LOW-crystallin aggregation for at least one hour. The
same concentrations of
-crystallin, however, had a
somewhat different chaperone effect on
N/C-crystallin. As seen in
Fig. 1h,
-crystallin is a less effective chaperone for
N/C-crystallin, and although the light scatter could be offset in
the presence of
-crystallin, precipitation nevertheless occurred as
a function of time, even when using increasing amounts of
-crystallin (4:1, w:w ratio, Fig. 1h).
The irreversible association of
LOW- to
-crystallin
at high protein concentrations (
10 mg/ml) was demonstrated for the first time by a combination of gel filtration, SDS gel electrophoresis (not shown), and SAXS as seen in Fig. 1, c and d.
After 30-min incubation at 60 °C, the gel filtration
-crystallin
peak is displaced toward higher molecular weight values, whereas the
LOW-crystallin peak disappears (Fig. 1c) in
agreement with the formation of soluble
/
LOW
aggregates. The association was easily shown by the SAXS experiments.
At room temperature the intensity curves for the
/
LOW
mixture corresponded to the sum of the intensity curves recorded for
- and
-crystallin alone. At 60 °C, the scattering of the
-crystallin alone cannot be recorded since the sample precipitates.
Yet, after incubation at 60 °C, the scattering at low angles for the
/
LOW mixture was higher than the scattering for the
high temperature form of
-crystallin indicating an increased average
molecular weight and therefore the association of
LOW- and
-crystallin, Fig. 1d).
The demonstration of an association between
- and
N/C-crystallin
was less straightforward, since at high protein concentration (10 mg/ml) the mixture precipitates as a function of time. Analysis of the
supernatant by gel filtration showed that part of the
-crystallin fraction still eluted at the same place (Fig. 1e), while by
SAXS no significant aggregation could be observed (Fig. 1f).
To clarify this point, SDS gel electrophoresis and Western blot
analysis of the "
-crystallin peak" were performed (Fig. 1,
i and k) using
C-crystallin and specific
antibody. The experiments demonstrated that
C/
-crystallin
complexes were present, but that at high protein concentration, once
associated, the
- and
C-crystallin co-precipitate rapidly.
The association between
- and
-crystallin was confirmed for
protein concentrations below 0.05 mg/ml by FRET. In these experiments mixtures of IAEDANS-labeled
S-crystallin (donor) and IAF-labeled
-crystallin (acceptor) were incubated in closed tubes at 66 °C for 10, 20, and 30 min. Emission spectra of each sample were recorded and compared with spectra at 25 °C before and after incubation. As
seen from Fig. 4a there is a progressive decrease in donor fluorescence (460 nm), indicating the
S/
-crystallin association at 66 °C with an estimated efficiency of transfer of about 0.15 after 30 min. Most importantly, the binding was irreversible, as the
decrease in donor fluorescence remained after the return to ambient
temperature. For comparison the emission of donor in the absence of
acceptor was unaffected by incubation at 66 °C (not shown). No
energy transfer could be detected for
S- and
-crystallin mixtures
incubated at 25 °C or 37 °C.
Native
-Crystallin Subunit Exchange--
The subunit exchange
in recombinant
A-crystallin had been demonstrated previously by FRET
(31, 33, 34). Here, we present data on the subunit exchange in native
calf
-crystallin, containing both
A (75%) and
B (25%)
subunits. Subunit exchange was studied at room temperature and 37 °C
when
-crystallin consists of 40-45 subunits on average, at 60 °C
and at 66 °C when the average number of subunits is 80-100 (Fig.
3, a and b). As can
be seen on Fig. 3b the time course of subunit exchange in
-crystallin is increasing with the increase in temperature and is
too fast to be reliably measured above 60 °C. Similar dependence on
temperature had been reported for
A-crystallin using other
fluorescent probes (34). The transfer rate constant at 37 °C was
estimated to be 6.33 × 10
4 s
1, in
agreement with the average value of 6.36 × 10
4
s
1 published for recombinant
A-crystallin (34). Also,
the energy transfer could be reversed by the addition of unlabeled
-crystallin (34). Therefore, it seems that the mechanism of subunit
exchange in native or recombinant crystallins is the same. We then
compared the subunit exchange at 37 °C before and after half an hour
incubation at 60 or 66 °C, i.e. after the heat-induced
-crystallin structural change. As can be seen from Fig.
3c the preincubation of
-crystallin at either 60 or
66 °C does not prevent subsequent subunit exchange at 37 °C. On
the contrary, the rate for subunit exchange at 37 °C for
heat-treated
-crystallin is significantly higher than for untreated
proteins. It therefore appears that the temperature-induced structural
changes lead to a more flexible and dynamic
-crystallin structure,
able of more rapid subunit exchange.

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Fig. 3.
Subunit exchange in
-crystallin. -IAEDANS (0.4 mg/ml) and
-IAF (0.4 mg/ml) were incubated at 60 °C for 30 min prior to
return to the ambient temperature. These samples were used for subunit
exchange at 37 °C: a, equal volumes of -IAEDANS and
-IAF were mixed, diluted to 0.05 mg/ml, and a series of spectra were
recorded ( ex = 335 nm) over a period of 2 h ( , 0 min; , 10 min; , 20 min; , 30 min; , 60 min; , 120 min).
b, -IAEDANS (0.4 mg/ml) and -IAF (0.4 mg/ml) were
preincubated each with an equal volume of LOW (0.4 mg/ml) at 60 °C for 30 min prior to return to ambient temperature.
Subunit exchange was monitored at 37 °C as described above for the
-crystallin alone (a). c, effect of
temperature on the subunit exchange. Subunit exchange between
-IAEDANS and -IAF at 15 °C ( ), 37 °C ( ), 37 °C
after preincubation at 60 °C for 30 min ( ), 37 °C after
preincubation at 66 °C for 30 min ( ), 60 °C ( ), 66 °C
(×). Spectra were recorded by following the time course of donor
fluorescence ( ex = 335 nm, em = 460 nm)
from equimolar mixture of -IAEDANS (donor) and -IAF (acceptor).
d, effect of preincubation with
LOW-crystallin at different ratios. -IAEDANS and
-IAF were incubated with LOW-crystallin at 1:0.5
( ), 1/1 ( ), and 1:2 ( ) w:w: ratios at 60 °C for 30 min.
Subunit exchange was carried at 37 °C. Consecutive emission spectra
( ex = 335 nm) were collected over 3 h, and the
intensities at maximum donor fluorescence ( em = 460 nm)
were plotted as a function of time.
|
|
Subunit Exchange and Chaperone Activity of
-Crystallin toward
Other Lens Crystallins by FRET--
LOW-Crystallin was
examined for the effect on
-crystallin subunit exchange. IAEDANS-
and IAF-labeled
-crystallins were heated at 60 °C for 30 min
either alone or in the presence of
LOW-crystallin. All
samples were returned to ambient temperature and stored overnight at
4 °C. Subunit exchange experiments were then carried out at
37 °C, and the extent of energy transfer was compared for mixtures
containing
-crystallins alone or preincubated with
LOW-crystallin. As can be seen in Fig. 3, a
and b, the extent of energy transfer, and therefore the rate
of subunit exchange, are significantly lower for
-crystallin
preincubated with
LOW-crystallin. The lowest spectra on
Fig. 3, a and b, represent the emission spectra
(excitation/emission 335/460) after two hours of subunit exchange. The estimated values for the efficiency of transfer (E) for
-crystallin alone and for
-crystallin
preincubated with
LOW-crystallin are 0.5 and 0.3, respectively. The decrease in the rate of subunit exchange was found
proportional to the
LOW/
-crystallin ratio (Fig.
3d). Such a response is in agreement with the formation of
LOW/
-crystallin hetero-complexes and could indicate
the presence of multiple binding sites and/or
-crystallin
association with
LOW-crystallin oligomers.
Differential Ability of
-Family Members to Inhibit
-Crystallin Subunit Exchange--
FRET was used to compare the
differential ability of the
-family members to associate with
-crystallin by following the rate of subunit exchange in the
presence of bound substrate. Fig. 4b presents the time course of
subunit exchange for
-crystallin preincubated for half an hour at
66 °C with the different
-crystallins at a w:w ratio 1:1. As can
be seen,
S is the most effective in slowing the
-crystallin
subunit exchange followed by h
Srec,
E,
A/F, then by
D and
by
B, which are much less efficient. In separate experiments,
-crystallin was preincubated at 66 °C with increasing amounts of
the different
-crystallins. For all
-crystallins studied the
inhibition of subunit exchange was dose-dependent (Fig. 4,
c-f). Ratios of
/
up to 4:1 (w:w) resulted in
corresponding increases of subunit exchange inhibition, suggesting
again the presence of multiple binding sites and/or association with
-crystallin oligomers. When repeated, on the same day or 1 week
later, the subunit exchange experiments consistently provided us with
the same results. Moreover, subunit exchange experiments using
previously assembled
/
complexes were reproducible over weeks. It
therefore appears that the
/
-crystallin complexes are
particularly stable.

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|
Fig. 4.
-crystallin subunit exchange in
the presence of different -crystallins.
a, the emission spectra of donor S-IAEDANS (0.05 mg/ml)
and acceptor -IAF (0.1 mg/ml) were recorded first at 25 °C using
ex = 335 nm ( ). Then temperature was changed to
66 °C and spectra recorded after 10- ( ), 20- ( ), and 30- ( )
min incubation. Finally, the temperature in cuvette was allowed to
return to 25 °C and spectra were recorded again ( ).
b-g, different -crystallins were compared for their
effect on the rate of -crystallin subunit exchange. -IAEDANS and
-IAF were preincubated at 66 °C (30 min) with equal volumes of
-crystallins solutions or buffer (control). Samples were allowed to
return to an ambient temperature before FRET experiments. Equal volumes
of preincubated protein solutions were mixed at 37 °C, and the time
course for the decrease in donor fluorescence was monitored at
em = 460 nm for 60 min. b, lanes from top
correspond to and different at 1:1 (w:w) ratios for S,
Srec, E, A-F, D, B, and alone.
c-g, lanes from top for and at different w:w
ratios: 1:4 ( ), 1:2 ( ), 1:1 ( ), 1:0.5 ( ), and 1:0 ( ).
c, and S; d, and Srec;
e, and E; f, and D; g,
and B. h, some weak but stable association is
recorded at 37 °C between individually heat-modified soluble lens
proteins. -IAEDANS, -IAF, and S were individually incubated at
66 °C for 30 min at 0.05 mg/ml each before the return to room
temperature. Donor -IAEDANS and acceptor -IAF were preincubated
further for 30 min at 37 °C with equal volumes of S ( ) or
buffer ( ) and used in subunit exchange experiment as described
above. -IAEDANS, -IAF, and LOW were treated the
same way except for the incubation temperature (60 °C). Subunit
exchange was recorded similarly in the presence ( ) or absence ( )
of LOW.
|
|
The Formation of Hetero-complexes at 37 °C Requires
"Activated" Partners--
In separate experiments, the subunit
exchange assay was used to check whether high temperature modified lens
proteins could form hetero-complexes at 37 °C. Proteins at
concentrations
0.05 mg/ml were heat-treated separately for 30 min at
low protein concentrations to avoid precipitation and returned to
ambient temperature prior to experiments. All proteins studied remained
soluble under these conditions. Subunit exchange was carried out at
37 °C as described in the legend of Fig. 4h. Data showed
that there was an inhibition of
-crystallin subunit exchange in the
presence of
LOW or of
S, therefore confirming the
/
LOW and the
/
S-crystallin association (Fig.
4h). However, the association was weaker, since the
inhibition of subunit exchange by both
LOW and
S was
smaller in comparison with data on Fig. 3, b and
d. Control experiments showed that the modification by
incubation of both
- and
LOW/
-crystallin was
required for the association to occur. Overall, these data support the
hypothesis that the chaperone activity of
-crystallin toward the
other lens crystallins requires an "activation," which in our case
is provided by the temperature induced structural transition, and that
this activated state is retained at lower temperature. Reciprocally,
only "aggregate competent"
LOW- or
-crystallin
can associate to activated
-crystallin, and the competent state may
be retained at physiological temperatures.
Effect of Bound Substrate on the Fluorescence of Labeled
-Crystallin--
Fluorescence characteristics of IAEDANS- and
IAF-labeled
-crystallin in the presence of different
LOW- or
-crystallins are in support of various
binding surfaces in
-crystallin. As can be seen from Fig.
5, a-f, there is a
differential response in both
-IAF and
-IAEDANS fluorescence upon
binding of
LOW,
S, or
D-crystallin. Clearly, the
environments around labeled residues were differently affected by
binding of each of the substrates, reflecting various binding
configurations of
-crystallin.

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Fig. 5.
Spectral changes in
-IAEDANS and -IAF induced
on the association with S,
D, or
LOW-crystallins. Spectral
characteristics of -IAF (a, c, d)
and -IAEDANS (b, d, f) change
differently upon temperature-induced association with various lens
proteins. -IAF and -IAEDANS were preincubated at 1:0.5 ( ) or
1:1 ( ) w:w ratios for 30 min with equal volumes of
LOW-crystallin at 60 °C (a, b),
S-crystallin at 66 °C (c, d), or
D-crystallin at 66 °C (e, f).
Spectra of -IAF and -IAEDANS with bound proteins were recorded
upon the return to ambient temperature using ex = 495 nm
and ex = 335 nm. g, the time course of
-IAF fluorescence ( ex = 495 nm, em = 512 nm) during the incubation at 66 °C alone ( ) or in the
presence of S (1:1, ). h, the time course of -IAF
fluorescence ( ex = 495 nm, em = 512 nm)
during the incubation at 60 °C alone ( ) or in the presence of
low (1:1, ).
|
|
Time Course of the Associations--
The formation of
hetero-complexes requires the activation of both the
-crystallin and
the
LOW/
substrates. The simultaneous occurrence of
these events at high temperatures is illustrated on Fig. 5,
g and h. Small aliquots of protein solutions
(either
alone or
:
LOW at 1:1, or
:
S at 1:1
mixtures) were introduced into preheated buffer inside the thermostated
cuvette at 60 or 66 °C, and the time course of
-IAF fluorescence
was recorded immediately after mixing. The upper lanes in
Fig. 5, g and h, show the
time-dependent increase in
-IAF fluorescence at 60 and 66 °C for
-crystallin alone, whereas the lower lanes show the
-IAF fluorescence in the presence of
LOW or
S. The
increase in
/IAF fluorescence as a function of time is in agreement
with the
-crystallin structural changes as mentioned above, whereas the inhibition in the presence of
LOW/
-crystallins
seems to indicate a rapid association of substrate with the outside
-crystallin subunits. It appears that the binding of
LOW- or
S-crystallin occurs rapidly, since an offset
in fluorescence of
-IAF starts immediately after the mixing of
proteins. All events therefore seem to occur simultaneously. It appears
that aggregate competent
LOW/
-crystallin forms
rapidly and start to associate with
-crystallin at the onset of its
structural changes.
 |
DISCUSSION |
As soon as it was demonstrated in vitro that
-crystallin could prevent
- or
-crystallin aggregation, it was
hypothesized that such a chaperone activity might be an important
factor to preserve in vivo lens transparency under various
stress conditions and in aging. The functional model behind was that
-crystallin was able to associate
- or
-crystallin at the
onset of its denaturation, thus preventing further unfolding and
precipitation, possibly protecting against cataract (3, 4, 54). To
better evaluate this model, we decided to compare the chaperone
properties of native calf
-crystallin toward other lens crystallins,
LOW and
, using various techniques, essentially SAXS
and FRET but also visible light absorption, gel filtration, gel
electrophoresis, Western blotting. The
LOW- or
-crystallin unfolding was induced by heat.
The first point addressed was the structural state of the various
partners during the chaperone reaction. It was known that
LOW- and
-crystallin start to unfold at 60 and
66 °C, respectively. From the changes in Trp fluorescence spectra,
we have shown that the unfolding remains limited. The
LOW-crystallin structure seems only slightly more open,
whereas changes in individual
-crystallin are even fainter. At the
low protein concentrations (about 25 µg/ml) used in fluorescence
experiments,
LOW- or
-crystallin aggregates after
unfolding, as shown by the appearance of a Rayleigh peak, but no
insoluble precipitate could be detected. Similar changes could not be
followed at high protein concentration, since the conformational
changes were immediately followed by crystallin aggregation and
precipitation. On the other hand, the data obtained by SAXS at high
protein concentrations provided us with quantitative parmeters for the
size changes of
-crystallin during and after heat treatment. The
changes include both a doubling in molecular weight accompanied
by the corresponding increase in size and more local changes like the
alteration in the environment around Cys residues demonstrated by
fluorescence. The changes were found irreversible on return to room
temperature and, whenever it was possible to check it, independent of
protein concentration. These results extend the previously published
observations using other physical techniques like EM (43). They are
also consistent with probing studies of recombinant
A- and
B-crystallin structure with
1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which indicated an increased exposure of hydrophobic surfaces after heat
induced conformational changes (55, 56).
The second point addressed was whether the protection against
precipitation was linked to crystallin-crystallin association. This
could easily be demonstrated for
LOW-crystallin, since
soluble
/
LOW-crystallin complexes were detected at
concentrations up to 20 mg/ml, by both gel filtration and SAXS. For
comparison, the detection at high protein concentration of
/
-crystallin complexes before precipitation could only be
demonstrated by Western blotting. At low concentration, however, the
presence of
/
-crystallin complexes was readily demonstrated by
FRET between labeled
- and
-crystallin (Fig. 4a).
Overall, the results obtained using SAXS and fluorescence confirmed
that the temperature-induced structural modifications were necessary
for hetero-complex formation between lens proteins. As a final result
it has been shown that separate preincubation at high temperature of
-crystallin and of
-crystallin may lead, upon mixing at 37 °C,
to the formation of
/
-crystallin complexes. Therefore, the whole
of these experiments emphasizes the importance of the structural
modifications of both
- and
LOW/
-crystallin for
the association to take place. On the contrary, the chaperone activity
of human recombinant
A- or
B-crystallin toward other partially
unfolded substrates, like insulin or lactalbumin, or thermally
denatured alcohol dehydrogenase or citrate synthase, or even
D-crystallin, yet denatured by singlet oxygen at 23 °C, does not
require the
-crystallin structural transition (9, 14, 16, 17, 20,
25, 29, 56, 57).
The third point addressed was the relationship between chaperone
properties and subunit exchange. Experiments on subunit exchange are
most easily performed by FRET (31, 33, 34). In the present study the
probes used were (5-IAF) and (1,5-IAEDANS). We have shown that the rate
of subunit exchange measured with these probes and calf
-crystallin
(which contains both
A (75%) and
B (25%)) was the same as the
one measured previously with other probes and recombinant
A (31).
The chaperone activity toward other lens proteins occurs, however, at
60 or 66 °C where the rate of exchange is too fast to be measured. A
modified subunit exchange assay was designed in which two populations
of
-crystallin, labeled either with IAF or IAEDANS, are separately
incubated at high temperature. The two populations are then brought
back to low temperature to stop the reaction, and the rate of exchange
is measured at 37 °C by recording the time-dependent
decrease in donor fluorescence after mixing of the two populations. The
assay demonstrated unambiguously that the temperature-modified
-crystallin retains its dynamic organization, i.e. can
still exchange subunits at 37 °C. In fact, it exchanges more rapidly
(Fig. 3), demonstrating that the temperature-induced structural
transition has altered the dynamic structure of
-crystallin. The
high temperature
-crystallin structure therefore appears to be an
"activated" structure and the activation to be linked to the
dynamic properties. The rate of subunit exchange is not concentration-dependent, which means that subunit exchange most likely occurs through a dissociation mechanism (34). One important implication is that the chaperone effect of
-crystallin
versus the other lens crystallins can then be evaluated by
following the rate of exchange at 37 °C after incubation at 60 or
66 °C for half an hour with the
LOW- or
-crystallin of interest. Fluorescence measurements are done at low
protein concentrations and under these conditions, the
/
LOW- and
/
-crystallin complexes remained soluble in solution and were stable for many days. This allowed us to
proceed with the comparison of the different
LOW- or
-crystallins. Such a comparison revealed considerable differences
between
-crystallins in their ability to interact with
-crystallin at high temperature, although
-crystallins are
homologous two-domain proteins that share from 50% (
S with the
others) up to 80% (
A-F) sequence identity. The more distant
S
and h
Srec were the most effective, with
h
Srec closely followed by
E and
A-F, and then by
D and
B. Despite their close sequence similarity,
-crystallins
are already known to present different physico-chemical properties, e.g.
B unfolds through stable intermediates (C-terminal
domain unfolded, N-terminal domain folded), while for
S no stable
intermediates could be detected under similar conditions (58, 59).
Phase separation temperatures are different: around 30 °C for
E
and
A-F, 4 °C for
D and
B, and no phase separation at all
for
S, either native or recombinant (60, 61). In the present study,
E and
A-F that have high phase separation temperatures
definitively appear more efficient in forming complexes than the low
phase separation temperature
D and
B. Yet, the differential
ability of the different
-crystallins to form
/
-crystallin
complexes does not seem to simply follow this well known
-crystallin
property (62, 63). Interestingly, the most efficient
-crystallin is
S, either native or recombinant, which is reminiscent of the observation that
S appears functionally special within the family. Indeed the
S, which in vivo is preferentially localized
in the lens cortex, could play a role in protecting against stress (47, 64). Yet the calf
S and the h
Srec, studied with calf
-crystallin as a chaperone, behave in a similar but not identical
way. The difference could reflect their different abundance and
possibly different specific functions in calf and in human. One may
therefore assume that the chaperone assay tests a combination of
stability and associative properties of the
-crystallins, which
could play a role in function. In separate experiments,
-crystallin
was preincubated with progressively increasing amounts of
LOW- or
-crystallins. All proteins studied were able
to interact with
-crystallin in a
concentration-dependent manner, the inhibition of exchange
increasing with increasing substrate-protein ratio and being clearly
different for the individual lens crystallins. Such data suggest the
presence of multiple binding sites on
-crystallin for
LOW- or
-crystallins and/or association with
LOW- or
-crystallin oligomers. Together with the
observation that the fluorescence of IAEDANS-labeled
-crystallin is
affected differently upon binding of the different
-crystallins, the
results argue for a variety of
LOW/
and
/
hetero-complexes. The binding stoichiometry could not, however, be
determined from such experiments. The whole of the experiments on
subunit exchange emphasizes that the dynamic organization of
-crystallins is an essential part of the chaperone effect and cannot
be dissociated from it. At 60 °C or 66 °C, a few events
simultaneously take place:
-crystallin increases in size,
LOW/
-crystallins bind to
-crystallin to form
complexes,
LOW/
-crystallins become aggregate
competent. Either the presence of activated
-crystallin in the
system permits to disrupt the
LOW/
-crystallin
aggregates, because of a competition for the binding sites, or the
LOW/
-crystallin association to
-crystallin occurs
at a faster rate. In any case, the fast subunit exchange process is
obviously an essential requirement for structural rearrangement and
protein incorporation. It is therefore reasonable to hypothesize, as
was done in other studies (65), that subunit exchange is a governing
mechanism in providing the necessary rearrangements to accommodate
substrate proteins.
Little is known about the structural characteristics of the
hetero-complexes. Yet, the inhibition of
/IAF fluorescence in the
presence of
LOW/
-crystallins would be consistent with
substrate coating around
-crystallin, rather than insertion between
subunits, because such a coating would conserve homotransfer between
subunits. Different
-crystallin peptides have been identified in
recent studies as functional elements in chaperone activity (66, 67). One of these peptides (residues 71-88 of
A-crystallin, (22, 68))
overlaps with putative substrate binding site of small heat shock
proteins (67). A second putative binding site was allocated to the
N-terminal part, involving aromatic residues (67). Recent studies using
mutated
-crystallin also demonstrated the involvement of the
C-terminal extension in its chaperone activity at 37 °C toward
insulin (69). However, all binding interfaces that have been proposed
were identified at low temperatures (25-37 °C) and may have
different properties at the temperatures at which lens heterocomplexes
are formed. For instance, according to the small heat shock
three-dimensional structures that have been determined (1, 2), one of
the proposed binding sites (residues 71-88) of
A-crystallin is
located on the surface but covered with the C-terminal extension of
another monomer. This site could become exposed during subunit exchange
via dissociation from oligomer and thus become available for
interactions with substrates.
In conclusion, we have shown that a high temperature stress induces a
partial unfolding of
LOW/
-crystallins and an
irreversible structural transition in
-crystallin resulting in a new
dynamic structure. The changes effect subunit exchange properties but do not abolish them. In this configuration,
-crystallin can form stable soluble complexes with aggregate competent
LOW-
or
-crystallins via multiple interactive surfaces over a wide range
of protein concentrations. Individual
-crystallins can be
differentiated by their ability to inhibit subunit exchange. Since the
chaperone activity of
-crystallin toward other lens proteins
required a heat-induced activation in vitro, the biological
relevance of such studies was questioned. We therefore wish to
emphasize that after our study, it rather appears that the most
important requirement for
-crystallin to act as a chaperone is that
of a dynamic structure. Given the
-crystallin structural plasticity,
our guess is that, in vivo,
-crystallin may be activated
in many ways to act as chaperone in a variety of stress
conditions and maintain lens transparency.
 |
FOOTNOTES |
*
This work was supported by the CNRS, the CNES, and the
European Community BIOMED contract "Aging Vision: Crystallins,
Visual Acuity, and Cataract."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.:
33-1-44-27-74-51; Fax: 33-1-44-27-37-85; E-mail:
Annette.Tardieu@lmcp.jussieu.fr.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M208157200
 |
ABBREVIATIONS |
The abbreviations used are:
FRET, fluorescence resonance energy transfer;
IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic
acid;
IAF, 5-(iodoacetamido)fluorescein;
SAXS, small angle x-ray
scattering;
EM, electron microscopy.
 |
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