α-Crystallin Modulates its Chaperone Activity by Varying the Exposed Surface

Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, Roma, RM, 00168 (Italy).
ChemBioChem (Impact Factor: 3.09). 11/2013; 14(17). DOI: 10.1002/cbic.201300447
Source: PubMed
ABSTRACT
The α-crystallin family of small heat shock proteins possesses chaperone activity in response to stress and is involved in several neurological, muscular, and ophthalmic pathologies. This family includes the vertebrate lens protein α-crystallin, associated with cataract disease. In this study, by combining small-angle X-ray and light scattering techniques, the structure and shape of α-crystallin was revealed in its native state and after a transition caused by heat stress. Below critical temperature (Tc ), α-crystallin appears as an ellipsoid with a central cavity; whereas at high temperatures the cavity almost disappears, and the protein rearranges its structure, increasing the solvent-exposed surface while retaining the ellipsoidal symmetry. Contextually, at Tc , α-crystallin chaperone binding shows an abrupt increase. By modelling the chaperone activity as the formation of a complex composed of α-crystallin and an aggregating substrate, it was demonstrated that the increase of α-crystallin-exposed surface is directly responsible for its gain in chaperone functionality.

Full-text

Available from: Massimiliano Papi
DOI: 10.1002/cbic.2013004 47
a-Crystallin Modulates its Chaperone Activity by Varying
the Exposed Surface
Valentina Palmieri, Giuseppe Maulucci, Alessando Maiorana, Massimiliano Papi, and
Marco De Spirito*
[a]
Introduction
a-Crystallin is the most abundant protein of eye lens fibre cells
and a member of the small heat shock proteins (sHSPs)
family.
[1]
sHSPs are highly conserved proteins, capable of chap-
erone activity under stress conditions, that is, able to bind un-
folded substrates in response to pH, temperature, or other
stress stimuli and prevent their aggregation and precipita-
tion.
[1,2]
a-Crystallin is isolated from eye lens as an 800 kDa
oligomeric complex made of aA and aB subunits (20 kDa) in
a 3:1 ratio.
[1]
These subunits are characterised by a 100-amino
acid core (common in the sHSP family) known as the a-crystal-
lin domain. This core is linked to N- and C-terminal extensions
(unconserved across the family). Extensions participate in the
association of the small subunits (or monomers) into large dy-
namic oligomers able to exchange components and mediate
recognition of client proteins.
[3]
The a-crystallin oligomeric
structure is still unknown, although several models have been
proposed thus far, such as three-layer,
[4,5]
micelle/bilayer-
like,
[6–8]
dodecahedral/octahedral,
[9,10]
pitted flexiball,
[11]
and
hollow sphere/bean with tentacles
[12]
structures.
The principle role of a-crystallin in eye lens is the mainte-
nance of tissue transparency.
[13]
To be transparent and allow
vision, the lenticular tissue is avascular and, during lens cells
maturation, organelles are removed, and the cytoplasm is
packed with short-range ordered, highly concentrated crystal-
lin proteins, which provide a transparent and refractive
medium.
[1]
Apart from a-crystallin lens cells, the cytoplasm con-
tains also b- and g-crystallins, small proteins closely related to
the chaperone but having only a structural function.
[1]
Due to
the loss of organelles, protein turnover is absent, and crystal-
lins have to maintain their native folding for the entire life of
the individual. As unfolding and/or post-translational modifica-
tions can occur, the capacity of a-crystallin to chaperone other
proteins represents the unique lens mechanism of protection
towards aggregate formation. This chaperone activity balances
the lack of protein synthesis and degradation in the lens and is
enhanced when stress makes crystallins prone to aggregate.
Impairment of a-crystallin activity, such as damage or genetic
mutation, or instability caused by b- and g-crystallins overload-
ing the chaperone, causes the appearance of aggregates and,
consequently, cataracts.
[13,14]
It follows that many studies have
focused on understanding the mechanisms responsible for the
regulation of chaperone activity. A variety of stress stimuli aris-
ing from small ligands, oxidation, pH, or temperature are
known to be regulators of a-crystallin chaperone activity.
[1,13,15]
For example, it has been observed that, upon thermal stress
beyond 30 8C, a-crystallin chaperone activity toward g-crystal-
lins increases, reaching the highest level at 558C, when g-crys-
tallin aggregation is totally prevented. Other studies identified
a critical temperature (T
c
=458C), above which oligomer struc-
tural modifications occur, including: 1) transition of the protein
to a larger structure,
[15–20]
2) increased exposure of hydro phobic
surfaces, 3) variation in subunit packing, and 4) subtle tertiary
structural changes.
[21–24]
It is widely accepted that a stress-
induced structural variation is a key mech anism commonly
adopted in the sHSPs family to regulate affinity to substrates
and avoid aberrant interactions.
[25]
Nevertheless, in the case of
a-crystallin, direct relationships have not been determined be-
tween the structural variations and an increase in chaperone
The a-crystallin family of small heat shock proteins possesses
chaperone activity in response to stress and is involved in sev-
eral neurological, muscular, and ophthalmic pathologies. This
family includes the vertebrate lens protein a-crystallin, associ-
ated with cataract disease. In this study, by combining small-
angle X-ray and light scattering techniques, the structure and
shape of a-crystallin was revealed in its native state and after
a transition caused by heat stress. Below critical temperature
(T
c
), a-crystallin appears as an ellipsoid with a central cavity;
whereas at high temperatures the cavity almost disappears,
and the protein rearranges its structure, increasing the solvent-
exposed surface while retaining the ellipsoidal symmetry. Con-
textually, at T
c
, a-crystallin chaperone binding shows an abrupt
increase. By modelling the chaperone activity as the formation
of a complex composed of a-crystallin and an aggregating
substrate, it was demonstrated that the increase of a-crystallin-
exposed surface is directly responsible for its gain in chaper-
one functionality.
[a] Dr. V. Palmieri,
+
Dr. G. Maulucci,
+
Dr. A. Maiorana, Dr. M. Papi,
Prof. M. De Spirito
Istituto di Fisica, Universit Cattolica del Sacro Cuore
Largo Francesco Vito 1, Roma, RM, 00168 (Italy)
E-mail: m.de spirito@rm.unicatt.it
[
+
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300447.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2362
CHEMBIOCHEM
FULL PAPERS
Page 1
activity at high temperatures, due to the lack of agreement on
a quaternary structure for the a-crystallin oligomer.
In this study, by combining small-angle X-ray scattering
(SAXS) and dynamic light scattering (DLS), we restored struc-
tures of native a-crystallin in physiological conditions and in
response to temperature stress. Among the activation mecha-
nisms, heat shock can be easily induced in SAXS and DLS
measurements, and protein response to this can be considered
a general mechanism of a-crystallin activation. Hence, we es-
tablished a structure–function relationship by modelling the
chaperone activity as the formation of a complex made of a-
crystallin and an aggregating substrate. We demonstrate how
a sharp transition in the amount of exposed surface is the
main structural feature responsible for the chaperone activity
enhancement of a-crystallin.
Results
a-Crystallin dimensions increase in response to temperature
stress
SAXS curves for a-crystallin (1.65 mg mL
1
) at temperatures
from 37 8Cto518C are shown in Figure 1A. Eventual denatura-
tion, aggregation, or interparticle interactions were excluded
(S1 and S2). Scattering curves do not display a well-defined
minimum or maximum and evidence an isoscattering point at
q=0.3 nm
1
. No large variation in the scattering intensity pro-
files occurs from 37 8Cto468 C(T
c
), whereas at higher tempera-
tures, an increase in scattering intensity at q = 0(I(0)) can be
observed. In our case, I(0) was proportional to the average mo-
lecular weight (MW) of scatterers; thus its increase at 49 and
518C is a hallmark of the formation of larger scattering ele-
ments. Accordingly, the radius of gyration (R
g
)ofa-crystallin,
estimated both with Guinier plot and pair distribution function
(P(r); Figure 1 B) increases from 6.64 nm at 378C to 8.13 nm at
518C. As expected from the observation of scattering profiles,
the R
g
of the protein is stable between 37 and 468C, although
a small decrease, more pronounced in P(r) analysis, is visible
between 37 and 408C. Above 46 8C, the protein has an abrupt
change in dimensions, and the R
g
displays a growth of 2 nm.
The temperature-dependent size increase agrees with those
already reported in literature.
[15–20]
The MW of a-crystallin
increases with tempe rature from 930 kDa (378C) to 1610 kDa
(518C; Figure 1 C). As the MW of a single subunit is 20 kDa, we
can roughly estimate that a-crystallin oligomers are composed
of 47 and 81 subunits at 37 and 51 8C, respectively. The MW,
analogously to the R
g
, remains stable between 37 and 468C.
From Kratky plots and P(r) functions, we determined oligomer
structural details. A sharp peak in the Kratky plot, typical for
globular and non-extended proteins (S2), was observed from
37 to 51 8C. P(r) distribution analysis confirmed this result;
along the temperature range, the bell-shaped P(r) distribution
rigidly shifts towards higher D
max
values, thus showing that the
protein almost preserves its shape while the dimensions in-
crease (S3).
The elliptical exposed surface of the a-crystallin oligomer
increases with temperature
SAXS data from the ab initio softwar e Dammin
[27]
gave tridi-
mensional models for a-crystallin at different temperatures. Tri-
dimensional reconstructions are shown in Figure 2 A, with each
structure representing a refined average of ten Dammin results
(see Experimental Section). A frontal, lateral, and basal view,
and a central section along the x-axes is shown. The influence
of a-crystallin polydispersity on reconstructions is small (S4),
and models can be considered good approximations for real
oligomeric shapes.
The protein maintains an elongated form with an elliptical
shape both before and after transition, but at 46 8C, there is
a decrease in an isometry which indicates a transition to
a more spherical symmetry. At 51 8C, the anisometry again
reaches a value comparable to that at 378C (Figure 2B). In the
internal structure of a-crystallin, a central cavity is visible at
Figure 1. Experimental scattering curves, B) R
g
, and C) MW of a-crystallin from 37 to 518C. A) Curves have similar profiles between 37 8C and 46 8C; beyond
this, an increase in MW at high temperatures is observed. Curves display an isoscattering point at q = 0.3 nm
1
. B) The R
g
of the protein was calculated with
Guinier approximation (
*
) and extrapolated from P(r)(
&
). The R
g
value is stable around 6.6 nm between 37 8C and 468C; beyond this, the temperature increas-
es abruptly and reaches a value of 8.2 nm at 51 8C. C) The MW has a similar behaviour compared to R
g
: it is stable at low temperatures and, after 468C, an in-
crease in mass causes the protein to a form that is doubled in MW (1650 kDa).
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2363
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 2
378C. In particular, the three major axes of the cavity have di-
mensions of 4.03.0 3.7 nm at 378C and 4.52.8 2.3 nm at
468C, whereas at 518C, we observed small empty spaces (di-
ameter<2 nm) that we did not consider as cavities owing to
the limit of resolution (S5). Protein surfaces of 3D reconstruc-
tions are reported in Figure 2 C. The exposed surface of a-crys-
tallin remains stable up to 468C and, at higher temperatures,
starts to increase. A direct comparison with the average
number of oligomers per temperature (N
olig
), inversely propor-
tional to the volume, allowed us to gain further insights on
this behaviour. Indeed, although the N
olig
of a-crystallin de-
creases, the protein balances this variation with increased ex-
position of surface.
As validation of the SAXS reconstructions, we compared hy-
drodynamic radii (R
H
)ofa-crystallin measured by DLS and
those computed theoretically from Dammin models.
[33]
From
DLS data, we determined the number weighted size distribu-
tion (P(i)) and recovered the R
H
of the protein in response to
temperature stress. The P(i) retains a single peak pattern at all
temperatures investigated (Figure 3 A). From 37 to 42 8C, the R
H
remains stable at 8 nm and then increases to 10.7 nm at 518C
(Figure 3B). The theoretical R
H
values are consistent with our
experimental DLS data and confirm the good quality of the
SAXS reconstructions (Figure 3B).
The behaviour of R
H
is clearly different from those observed
for both R
g
(Figure 1B) and MW (Figure 1C). The analysis of the
shape factor s (i.e., the R
g
/R
H
ratio)
[38]
allowed us to gain details
of the protein shape (Figure 2 B): s goes from a value of 0.84
at 378C to 0.73 at 45 8C, then increases to 0.8 at 518C. As s is
0.7 for a sphere, the protein appears to be elongated at 378C,
acquires a more spherical shape at 46 8C, and returns to an
elongated shape at 51 8C. The shape factor trend with temper-
ature supports the anisometry analysis of our reconstructions.
a-Crystallin suppresses g-crystallin aggregation
The aggregation of g-crystallin in the absence or presence of
a-crystallin at 37 and at 498C was measured (Figure 4 A and B,
respectively). The backscattering intensity of thermally aggre-
gated g-crystallin was measured by using DLS. In Figure 4A,
the first phases of aggregation of g-crystallin at 378C are re-
ported in the absence and presence of a-crystallin. The aggre-
gation proceeds at a slower rate when a-crystallin is present.
The same effect is more pronounced at temperatures above
the T
c
(Figure 4B shows the effect at T=49 8C).
Discussion
a-Crystallin is a small chaperone involved in the suppression of
aggregate appearance in cataracts.
[1]
Its functional dynamics
were characterised by using environmental stress stimuli, not
necessarily within physiological ranges, which are known to
Figure 2. Structural reconstruction and hydrodynamic properties of a-crystallin A) Reconstruction of the a-crystallin structure obtained from SAXS experi-
ments at different temperatures. The 3D models in this figure represent a refined average obtained with Damaver. For each model, there is a frontal (first
line), lateral (second line), and basal (third line) view, whereas the last line represents a central section along the x-axis. Beyond 498C, the protein has an
abrupt increase in dimensions and retains an elliptical shape both at 37 and 518C. At 46 8C, the structure appears slightly more spherical. B) The shape factor
s (
~
) and the anisometry (
&
) are plotted. Both values indicate a transition toward a more spherical form at 46 8C, but, as values at 37 and 51 8C are similar,
the protein maintains an elliptical symmetry before and after the transition. C) The surface (
&
) of the protein continues to increase with temperature at 46 8 C,
whereas the global N
olig
(
~
) decreases at high temperatures, as it is in inverse proportion to the volume of the particle. This indicates that, although the N
olig
decreases, the protein balances this trend with an increase in the exposed surface.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2364
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 3
improve chaperone activity. Heating is one of the stress stimuli
most widely adopted to trigger a-crystallin chaperone efficien-
cy, although this protein also responds to small-ligand binding,
pH, or redox variations.
[1,2,14]
It is well established that chaperone efficiency of the protein
is enhanced at temperatures higher than 378C.
[39,40]
As an ex-
ample, at low temperature, a-crystallin has very little effect on
the photo-aggregation of g-crystallin. On the other hand, the
protection towards g-crystallin aggregation is complete when
a-crystallin is subjected to heat shock. Interestingly, the same
effect was observed with other model systems, such as DTT-
induced aggregation of insulin and refolding-induced aggrega-
tion of b-crystallin,
[22]
suggesting that a-crystallin adopts the
same strategy to inhibit aggregation, aside from the stress
stimulus.
In this study, the effect of temperature on oligomers of a-
crystallin was analysed in order to obtain a model that explains
the relationship between chaperone activity and protein struc-
ture while increasing the temperature stress. At high tempera-
tures, the dimensions of the a-crystallin oligomer increase and
the protein doubles its mass, from 930 kDa at 378Cto
1610 kDa at 518C. Below 468C, the R
g
is stable; above 468C,
a steep growth is observed, in agreement with those already
reported.
[15–19]
Although the dimensions of a-crystallin increase,
the ellipsoid shape is conserved both at 37 and 51 8C.
Although the shape is conserved, two major structural differ-
ences, one in internal structure and the other in exposed sur-
face, emerge. Firstly, the protein possesses an inner cavity with
a diameter around 3 nm at 37 8C. At 51 8C, when a-crystallin
displays the maximum chaperone efficiency, the cavity disap-
pears from structures. The presence of a cavity in native a-crys-
tallin has been suggested in many models and,
[6–8,12,41]
in trans-
mission electron microscopy micrographs, regions of lower
protein density were observed.
[41]
In the sHSPs family, the pres-
ence of an internal cavity is a common feature,
[25]
and it has
been proposed that the chaperone activity of these proteins
consists of binding substrates in the internal space. Alternative-
ly, it has been suggested that the sHSPs might accommodate
substrate proteins on the solvent-exposed face, where multiple
hydrophobic sites would allow coating by their clients.
[18,41]
We
detected a cavity of about 15 nm
3
at 378C (S5), large enough
to accommodate small proteins in the a-crystallin oligomer.
Unfortunately, as it has been demonstrated that single a-crys-
tallin oligomer can bind up to seven substrate units and can
interact with large enzymes, we excluded the cavity as the
lone binding site.
[7,42,43]
Furthermore, disappearance of the
inner cavity at 518C does not explain the increase in chaper-
one function at this temperature. Recent evidence suggests
that a-crystallin might well have different chaperone mecha-
nisms depending on stress conditions or substrate, and the ex-
istence of both low affinity (on the surface) and high affinity
(in the cavity) binding sites in the same oligomer has been
hypothesised.
[44,45]
Hence, we focused our analysis on the possibility that a-
crystallin chaperone activity depends directly on the surface
binding properties. As we found that the a-crystallin exposed
surface undergoes an abrupt increase at T
c
, we suggest that
a quaternary transition is the mechanism adopted by the pro-
tein to enhance its chapero ne activity during stress.
Chaperone activity is generally quantified by measuring
chaperone efficiency (C.E.),
[40]
that is, the percentage of protec-
tion of a-crystallin towards denat ured substrates, calculated
according to the expression:
C:E:
t¼t
0
¼ 100
I
g
ðt
0
ÞI
ga
ðt
0
Þ
I
g
ðt
0
Þ
ð1Þ
where I
g
(t
0
) is the intensity of scattered light for the substrate,
and I
ga
(t
0
) is the scattering intensity of the same substrate in
the presence of a-crystallin at time t
0
.
As previously observed, C.E. values, calculated at t
0
= 80 min,
increases non-linearly with temperature, reaching the highest
value at 518C when a-crystallin totally prevents thermal aggre-
gation of g-crystallin (Figure 4 C). Unfortunately, C.E. depends
not only on the chaperone activity but also on parameters
such as the initial monomer concentration and rate of aggre-
gation: aggregate mass, size, shape, etc.
[36]
Therefore, possible
Figure 3. Hydrodynamic properties of a-crystallin at different temperatures
A) In P(i) distributions, a-crystallin retains a single peak at 378C(c), at
458C(g) and at 51 8C(d), while moving toward higher values of R
H
.
B) The R
H
of the protein was calculated with DLS by using CONTIN (
&
) and
compared to values extrapolated with Hydropro on Dammin reconstructions
(
~
). Both analyses give similar results: the R
H
of a-crystallin starts to increase
at 46 8C, going from 8.1 nm at 37 8C to 10.7 nm at 51 8C. The red line is
a polynomial fit of DLS results used for extrapolation of values at different
temperatures.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2365
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 4
modifications of these parameters, occurring over the time
course of aggregation and depending on the specific sub-
strate, affect the C.E. and lead to an incorrect quantification of
the chaperone activity.
To monitor the chaperone activity, instead of intensity, we
used the chaperone rate constant k
C
, which establishes a link-
age between structural and functional features independently
for the substrate and on the aggregation modalities. The k
C
value can be indirectly measured by modelling the initial
phases of aggregation process of g-crystallin. We assume that,
in the first phases of aggregation, g-crystallin forms dimers at
a rate constant of k
A
unless a-crystallin sequesters g-crystallin
by forming an ag-complex at a rate constant of k
C
(Figure 4E).
Accordingly, the backscatt ered intensity can be written as:
I
bs
K½m
2
a
N
a
þ m
2
ag
N
ag
þ m
2
g
ðN
g
þ 4N
2g
Þ
ð2Þ
where K is an optical instrumental constant, the couples (m
a
,
N
a
), (m
ag
, N
ag
), and (m
g
, N
g
) represent the mass and the number
of a-crys tallin, the ag-complex, and g-crystallin, respectively. In
the first steps of aggregation, the particles are much smaller
than the scattering length (qR
ag
! 1), and the form factors of
the three particles can be assumed to be equal to one. When
m
a
@ m
g
, m
a
~m
ag
, Equation (1) becomes:
I
bs
K½m
2
a
ðN
a
þ N
ag
Þþm
2
g
ðN
g
þ 4N
2g
Þ
ð3Þ
and when N
a
+ N
ag
is constant
I
bs
A þ K
0
ðN
g
þ 4N
2g
Þ
ð4Þ
where A and K are auxiliary constants. The time evolution of
N
g
and N
2g
can be modelled according to the following differ-
ential equations:
dN
g
dt
¼k
C
N
g
k
A
N
2
g
dN
2g
dt
¼ k
A
N
2
g
8
<
:
ð5Þ
Integration of this uncoupled system yields expressions for N
g
and N
2g
Figure 4. Suppression of g-crystallin aggregation due to a-crystallin chaperone action, comparison between chaperone efficiency (C.E.) and k
C,
and models for
a- and g-crystallins interaction. The aggregation of g-crystallin in the absence (
~
) or presence (
^
)ofa-crystallin at A) 37 8 C and B) 498C is shown. C) From the
intensity of aggregation, the C.E. (e) was measured and plotted against temperature. The dependence is nonlinear, with continuous growth until the protein
reaches about 90 % of e at 51 8C. On the other hand, the k
C
between a- and g-crystallin has a sharp transition at 46 8C, when it abruptly starts to increase.
D) The relationship between the surface increase of a crystallin with temperature and chaperone efficiency was plotted. The relationship between e and sur-
face has a nonlinear depend ence, whereas plotting the exposed area and k
C
shows a linear correlation. E) g-Crystallin aggregation begins with unfolding of g-
crystallin to forms dimers, the building blocks for aggregates. In the presence of a-crystallin, unfolded substrate is sequestered and, before aggregation, an
ag complex is formed with k
C
.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2366
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 5
N
g
¼ N
g0
k
C
k
C
e
k
C
t
þ k
A
N
g0
ðe
k
C
t
1Þ
ð6Þ
N
2g
¼
k
c
k
A
ðk
c
t
N
g0
k
A
k
c
e
k
c
t
þ N
g0
k
A
ðe
k
c
t
1Þ
log ðk
c
e
k
c
t
þ N
g0
k
A
ðe
k
c
t
1ÞÞÞ þ
N
g0
k
A
þ k
c
logðk
c
Þ
k
A
ð7Þ
Substituting Equations (13) and (14) (Experimental Section)
for Equation (11), we fitted the theoretical expression of I
bs
to
the experimental data, yielding the k
C
and k
A
values reported
in Table 1. In Figure 4C, we report k
C
and C.E. values at differ-
ent temperatures. Although a sharp transition, occurring at
468C, can be observed for k
C
values (solid squares), this is not
the case for C.E. values (open diamonds), which show a much
smoother transition. This discrepancy is a clear mark of the
modifications of shape and size of g-crystallin aggregates oc-
curring in the time course of the aggregation process affecting
C.E. Indeed, the scattered intensity is affected by both en-
hancement of the chaperone constant and by a number of
structural and kinetic parameters [Eq. (8)], which are difficult to
calculate.
[36]
As enhancement of the binding cap acity occurs together
with the surface modification of a-crystallin oligomers (Fig-
ure 2 C), we correlated the oligomer surface with both the
chaperone rate constant and the C.E. (Figure 4 D). As expected,
whereas the C.E. shows a non-linear increase with the surface,
a clear relationship between k
C
and S can be observed (R=
0.987). This evidence suggests, furthermore, that the number
of binding sites increases with the protein surface and leads to
enhancement of the chaperone activity. On the other hand,
the cavity cannot be directly related to k
C
. Overall, this study
suggests that, upon heat stress, a-crystallin undergoes shape
remodelling that affects its chaperone activity.
The chaperone activity of this protein, inhibiting aggrega-
tion, is crucial in many pathologies. The substrate specificity of
a-crystallin is rather broad, and chaperone activity toward vari-
ous lenticular and non-lenticular proteins in response to physi-
cal and physiological stresses such as heat, UV irradiation,
chemical stress, oxidative stress, and glycation has been ob-
served.
[46,47]
It is generally accepted that a-crystallin, irrespective of the
involvement of a specific portion of the sequence mediating
the substrate binding, suppresses aggregation through the
interaction between hydrophobic patches on its surface and
exposed hydrophobic sites of partially unfolded substrate
protein. Essentially, hydrophobic sites on the surface of the
protein, either present in the native states or exposed after
structural modification of the protein, associate with a variety
of partially denatured proteins in a substrate dependent
manner.
[18,46]
The strongest supporting evidence for the involvement of
hydrophobicity in chaperone function comes from tempera-
ture-dependent experiments.
[46]
Upon heating, a-crystallin un-
dergoes structural changes, resulting in increased exposure of
additional hydrophobic sites associated with increased chaper-
one activity.
Conclusions
In this study, we confirm a leading role fo r the surface in rela-
tion to functionality of the a-crystallin protein. We suggest the
existence of binding sites distributed on the protein surface
that increase with temperature. This hypothesis is further con-
firmed by subunit exchange studies;
[18]
this demonstrated that,
in the presence of b- and g-crystallins, an ab/ag-complex is
formed, and the exchange of a-crystallin subunits is inhibited
because of a substrate coating around the oligomer. Moreover,
a decrease in subunit exchange rate was found to be propor-
tional to the substrate-to-a-crystallin ratio, thus suggesting
multiple binding sites on the surface. This behaviour necessi-
tates a-crystallin temperature-induced structural modification
and hence confirms our findings on surface exposition with
temperature transition.
In addition to the role played by the a-crystallin surface, the
existence of a chaperone site residing in a central cavity in the
oligomer has been proposed.
[41]
Evidence for target proteins
binding inside the central cavity of a-crystallin comes from
electron microscopy (EM) and small-angle neutron scattering
data.
[48,49]
In both cases, on the basis of the radius of gyration
of the complex, it was suggested that g-crystallin, denatured
by means of chemical or thermal processes, binds the central
cavity of an a-crystallin oligomer both in its native state and
after transition. Our 3D reconstructions, however, show an
empty region in the central region of a-crystallin. Furthermore,
a functional role of the cavity has been ruled out; indeed: 1) it
almost disappears when the chaperone activity is enhanced at
high temperature and 2) the volume enclosed inside the cavity
is too small to account for the amount and/or the dimensions
of proteins that a-crystallin can bind. As a matter of fact, it is
interesting to note that a-crystallin prevents the thermal ag-
gregation of several proteins at tenfold lesser concentrations
than the target substrate.
[39]
Our results, while representing a step forward in the under-
standing of the chaperone mechanism of a-crystallin, specifi-
cally in the light of prevention and treatment of cataracts with-
out surgery, furnish relevant improvements in the area of the
misfolding pathologies
[50]
in which a-crystallin chaperone func-
tion failure is believed to have a role. Indeed, it is well known
that a-crystallin chaperone activit y is also relevant in many tis-
Table 1. k
A
and k
C
values at different temperatures.
[a]
T [8C] k
A
[m
1
s
1
] k
C
[m
2
s
1
]
37 (1.990.12) 10
4
(2.430.20) 10
4
43 (2.610.36) 10
4
(3.640.13) 10
4
46 (3.230.49) 10
4
(4.860.16) 10
4
49 (7.791.4) 10
3
(1.390.08) 10
2
51 (2.871.5) 10
2
(2.950.14) 10
2
[a] k
A
and k
C
represent, respectively, the rate constants for formation of g-
crystallin dimer or ag complex as measured by DLS experiments.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2367
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 6
sues, other than eye lens, and it is therefore quite reasonable
that a strategy to enhance its chaperone activity would be as
well.
Experimental Section
a-Crystallin and g-crystallin sample preparation: Bovine eye lens
a-crystallin was prepared as described previously.
[17]
Briefly, the a-
crystallin fractions were suspended in 10 mm Tris·HCl buffer,
pH 7.4. The purified protein was divided into aliquots and kept in
the same buffer at 208C until use. Just before experiments, the
samples were thawed and centrifuged at 5000 g (Eppendorf 5418)
for 30 min to discard supramolecular aggregates. The supernatant
was filtered through a 0.22 mm Millipore low-retention filter directly
into the measuring cuvette. Protein concentration was determined
by using an absorption coefficient of A (0.1 % 1 cm) =0.81 at
280 nm. SAXS experiments at different temperatures were per-
formed, waiting 30 min for protein equilibrium before the acqui-
sition of the data. g-Crystallin (Sigma–Aldrich) was suspended
in 10 mm Tris·HCl buffer, pH 7.4, at a final concentration of
0.2 mg mL
1
. For aggregation experiments, g-crystallin alone or to-
gether with a-crystallin was exposed to different temperatures
during DLS experiments.
Small-angle X-ray scattering (SAXS): SAXS data were collected by
using the ID14 high brilliance beamline at the European Synchro-
tron Radiation Facility (ESRF) of Grenoble. For accurate subtraction
of the scattering deriving from cell and solvent, buffer scattering
was measured. Two-dimensional SAXS patterns were azimuthally
averaged in order to obtain the scattered intensity I(q) as a function
of the X-ray-exchanged momentum (q) in the range from 0.05 to
4.27 nm
1
. Data were converted to absolute intensity values by
normalising to the scattering signal of water. Each acquisition was
averaged over 1 s in order to avoid sample damage, with measure-
ments successively repeated at different positions on each sample
to augment the number of statistics for analysis. Measurements
were performed at temperatures between 37 and 518C and in
a concentration range from 0.78 to 12.5 mgmL
1
. Software pack-
age ATSAS 2.4 was used for data analysis.
[26]
For evaluation of data
quality, protein unfolding was evaluated from the Kratky plot,
[27,28]
obtained by plotting q
2
I(q) versus q, where an increase of q
2
I(q)at
high q values indicates unfolding. Global structural parameters
were extracted from data by using program PRIMUS.
[26]
In particu-
lar, as the forward scattering intensity I(0) is proportional to the
MW of the protein, the mass of a-crystallin was calculated by com-
parison with intensity of a bovine serum albumin reference. Parti-
cle size was estimated from the radius of gyration (R
g
). The values
of I(0) and R
g
were derived from experimental scattering intensities
I(q) by using the Guinier approximation,
[28]
IðqÞ¼Ið0Þexpðq
2
g
2
=3Þ
ð8Þ
R
g
and I(0) were respectively inferred from the slope and the inter-
cept of the linear fit ln( I(q )) versus q
2
in the range 0.8R
g
< q<
1.30215R
g
.
[29]
From the linear dependence of log(I(q)) versus q
2
, the
sample aggregation was tested. Estimation of D
max
, the maximum
particle diameter, was obtained through the pair-distance distribu-
tion function P(r),
Ið0Þ¼4p
Z
D
max
0
PðrÞdr
ð9Þ
calculated by the indirect Fourier transform program GNOM.
[26,27,30]
GNOM extrapolations of the values of I(0) and R
g
were compared
with Guinier data.
SAXS protein structure reconstruction and characterisation:
Low-resolution protein envelopes were restored from experimental
data by using ab initio shape determination program Dammin,
based on a simulated annealing procedure.
[27]
Dammin uses input
files processed by GNOM. Hence, P(r) was evaluated by cutting
data to a maximum s value of 1.6 nm
1
. The D
max
was automatically
read by Dammin from GNOM files, the radius of the dummy atoms
used was 5.6 , and no symmetry was constrained; default settings
were retained for other parameters. The quality of the fit was eval-
uated with the c
2
of Dammin, which is the difference between
experimental and model scattering patterns. To validate the repro-
ducibility of Dammin results, each fit (per temperature) was per-
formed ten times, and separated reconstructions were compared
and aligned with Supcomb and averaged by using Damaver.
[31]
The
tridimensional structures generated by Damaver were then used as
starting points for Dammin for a refinement of the averaged data.
Anisometry and total excluded volume (TEV) of the structures were
calculated by Dammin; more precisely, the anisometry of a particle
was computed as
Anisometry ¼
ðPr Axe1 Pr Axe3Þ
ðPr Axe1 þ Pr Axe3Þ
ð10Þ
Given a Dammin PDB, the principal axes of inertia are computed
as eigenvalues of the inertia tensor. The eigenvalues are sorted in
descending order: PrAxe1 (longest), PrAxe2, PrAxe3 (shortest). The
anisometry ranges from zero for a sphere to unity for a needle.
TEV is computed by multiplying the volume of a single dummy
atom by the total number of dummy atoms divided by 0.74. The
number of a-crystallin oligomers in the sample was calculated by
considering it as an inverse proportion of the volume of the pro-
tein. UCSF Chimera software was used for surface calculation from
volumetric map data of 3D structures.
[32]
Theoretical hydrodynamic
properties of tridimensional structures were calculated with the
program Hydropro, which uses a bead shell model procedure to
simulate hydrodynamic behaviour of proteins, given their atomic
structure.
[33]
Polydispersity analysis: To evaluate how polydispersity can influ-
ence Dammin results, 3D models from SAXS curves of spheres with
different size distribution functions, that is, progressively increased
polydispersity, were simulated. SASfit software was used to simu-
late SAXS curves, and the volume distribution function D(R)ofa-
crystallin,
[34]
considered as a polydisperse system of spheres, was
calculated with GNOM. D(R) is defined as
DðRÞ¼
4
=
3
pR
3
NðRÞ
ð11Þ
where R is the sphere radius and N(R) the relative number of parti-
cles with this radius in the system. D(R) was fitted with a LogNormal
distribution with equation
f
x
ðx; m; sÞ¼
1
xs
ffiffiffiffiffi
2p
p
e
ðln xmÞ
2
2s
2
ð12Þ
and parameters of x, m, and s for a-crystallin data were obtained.
Then, I(q) values were obtained for spheres having the size distri-
bution of the protein or having different grades of polydispersity
(i.e., different s values). Finally, simulated I(q) values were used to
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2368
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 7
perform Dammin reconstruction to evaluate program sensitiveness
to polydispersity.
Dynamic light scattering: Dynamic light scattering (DLS) was used
to obtain the hydrodynamic properties of a-crystallin in response
to temperature variations. DLS measurements were performed by
using a commercial computer-interfaced scattering instrument
ALV/SLS-5000 system (ALV, Langen, Germany) equipped with
a 50 mW HeNe laser operating at 632.8 nm. The autocorrelation
function of the photopulses was performed by a 256-channel digi-
tal correlator (ALV-5000). Counts per second were used to measure
the scattered intensity during the aggregation. The DLS technique
measures the intensity autocorrelation function
g
2
ðtÞ¼hIðtÞIðt þ tÞi=hIi
2
ð13Þ
where t is the lag time, and brackets represent the ensemble aver-
age.
[35]
The g
2
(t) value can be related to the field autocorrelation
function g
1
(t) through the Siegert relation g
2
(t)= 1+bg
2
1
(t), where
b is an instrumental constant (in our setup b =1). The mathemati-
cal form of g
1
(t) depends on the physical properties of the investi-
gated system. For monodisperse particles, the electric field auto-
correlation function decays exponentially following g
1
(t)=e
Gt
,
where the decay rate G depends on the particle translational diffu-
sion coefficient according to G = Dq
2
. For a polydisperse sample,
g
1
(t) is more complex than a single exponential. Under this condi-
tion, the derivative of g
1
measures the intensity-weighted average
decay rate of the clusters:
G
I
¼
d ln g
1
ðtÞ
dt
j
t¼0
< ZS < ð14Þ
To determine
G
I
experimentally, we fitted the logarithm of the
measured autocorrelation function g
1
to a second-order poly-
nomial, according to the cumulant expansion:
[28]
ln½g
1
ðtÞ ¼ G
1
t þ
1
2
G
1
t
2
þ oðt
3
Þ
ð15Þ
where we assumed G
I
= G
1
. In aggregating systems, because of
cluster–mass polydispersity, what we actually measure is an aver-
age effective diffusion coefficient that can be expressed as:
D
I
¼
G
q
2
¼
R
1
0
p
I
ðrÞ
GðrÞ
q
2
dr
R
1
0
p
I
ðrÞdr
ð16Þ
where p
I
(r) is the normalised intensity-weighted radius distribution
function describing the distribution of the fraction of the intensity
scattered by a particle of hydrodynamic radius r and decay rate
G(1), given by:
Gð1Þ¼kTq
2
=6phr
ð17Þ
where h is the water viscosity and k the Boltzmann constant. The
intensity-weighted average effective hydrodynamic radius r
¯
1
can
be obtained from the Stokes–Einstein equation:
r
1
¼ kT=6ph
D
1
ð18Þ
The complete distribution of decay rates can also be recovered by
introducing the equation:
[36]
g
I
ðtÞ¼
Z
1
0
p
I
ðrÞe
GðrÞ
ð19Þ
Recovery of the p
I
(r) distribution, a classically ill-conceived problem,
can be obtained by performing a regularised Laplace inversion of
the intensity autocorrelation function by using the software
Contin.
[37]
Acknowledgements
SAXS experiments were performed on the ID14 beamline at the
European Synchrotron Radiation Facility (ESRF), Grenoble, France.
We are grateful to Louiza Zerrad and Petra Pernot at ESRF for
providing assistance in using the beamline. DLS data reported in
this paper were obtained at the LABCEMI (Laboratorio Centraliz-
zato di Microscopia Ottica ed Elettronica) facility of the Universi-
t Cattolica del S. Cuore, Rome (Italy).
Keywords: alpha-crystallin · chaperone proteins · heat shock
proteins · protein structures · small-angle X-ray scattering
[1] H. Bloemendal, W. de Jong, R. Jaenicke, N. H. Lubsen, C. Slingsby, Prog.
Biophys. Mol. Biol. 2004, 86, 407 485.
[2] F. Narberhaus, Microbiol. Mol. Biol. Rev. 2002, 66, 64 93.
[3] S. J. Eyles, L. M. Gierasch, Proc. Natl. Acad. Sci. USA 2010, 107, 2727
2728.
[4] R. J. Siezen, H. J. Hoenders, Eur. J. Biochem. 1979, 96, 431 440.
[5] A. Tardieu, D. Laporte, P. Licinio, B. Krop, M. Delaye, J. Mol. Biol. 1986,
192, 711 724.
[6] M. T. Walsh, A. C. Sen, B. Chakrabarti, J. Biol. Chem. 1991, 266, 20079
20084.
[7] J. A. Carver, J. A. Aquilina, R. J. Truscott, Exp. Eye Res. 1994, 59, 231 234.
[8] R. C. Augusteyn, A. Stevens, Prog. Polym. Sci. 1998, 23, 375 413.
[9] P. J. Groenen, K. B. Merck, W. W. de Jong, H. Bloemendal, Eur. J. Biochem.
1994, 225, 1 19.
[10] A. M. Morris, J. A. Aquilina, Proteins Struct. Funct. Bioinf. 2010, 78, 2546
2553.
[11] R. H. Smulders, M. A. van Boekel, W. W. de Jong, Int. J. Biol. Macromol.
1998, 22, 187 196.
[12] J. Vanhoudt, S. Abgar, T. Aerts, J. Clauwaert, Eur. J. Biochem. 2000, 267,
3848 3858.
[13] C. Cheng, C. H. Xia, Q. Huang, L. Ding, J. Horwitz, X. Gong, J. Biol. Chem.
2010, 285, 41187 41193.
[14] J. J. Harding, Ageing Res. Rev. 2002, 1, 465 479.
[15] M. R. Burgio, P. M. Bennett, J. F. Koretz, Mol. Vision 2001, 7, 228 233.
[16] F. Skouri-Panet, S. Quevillon-Cher uel, M. Michiel, A. Tardieu, S. Finet, Bio-
chim. Biophys. Acta Proteins Proteomics 2006, 1764
, 372 383.
[17] G. Maulucci, M. Papi, G. Arcovito, M. De Spirito, PLoS One 2011, 6,
e18906.
[18] T. Putilina, F. Skouri-P anet, K. Prat, N. H. Lubsen, A. Tardieu, J. Biol. Chem.
2003, 278, 13747 13756.
[19] J. Vanhoudt, S. Abgar, T. Aerts, J. Clauwaert, Biochemistry 2000, 39,
4483 4492.
[20] J. W. Regini, J. G. Grossmann, M. R. Burgio, N. S. Malik, J. F. Koretz, S. A.
Hodson, G. F. Elliott, J. Mol. Biol. 2004, 336, 1185 1194.
[21] M. Haslbeck, T. Franzmann, D. Weinfurtner, J. Buchner, Nat. Struct. Mol.
Biol. 2005, 12 , 842 846.
[22] C. M. Rao, T. Ramakrishna, S. Y. Pasta, B. Raman, Stress Response: Mol.
Biol. Approach 2006, 37, 93 134.
[23] K. P. Das, W. K. Surewicz, FEBS Lett. 1995, 369, 321 325.
[24] G. Maulucci, M. De Spirito, G. Arcovito, M. Papi, PLoS One 2012, 7,
e30705.
[25] H. S. McHaourab, J. A. Godar, P. L. Stewart, Biochemistry 2009, 48, 3828
3837.
[26] P. V. Konarev, M. V. Petoukhov, V. V. Volkov, D. I. Svergun, J. Appl. Crystal-
logr. 2006, 39, 277 286.
[27] D. I. Svergun, M. H. J. Koch, Rep. Prog. Phys. 2003, 66, 1735 1782.
[28] C. D. Putnam, M. Hammel, G. L. Hura, J. A. Tainer, Q. Rev. Biophys. 2007,
40, 191 285.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2369
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 8
[29] H. D. Mertens, D. I. Svergun, J. Struct. Biol. 2010, 172, 128 141.
[30] G. Ciasca, M. Papi, M. Chiarpotto, M. Rodio, G. Campi, C. Rossi, P.
De Sole, A. Bianconi, Appl. Phys. Lett. 2012, 100, 073703.
[31] V. V. Volkova, D. I. Svergun, J. Appl. Crystallogr. 2003, 36, 860 864.
[32] E. F. Pettersen, T. D. Goddard, C. C. Huan g, G. S. Couch, D. M. Greenblatt,
E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004, 25, 1605 1612.
[33] A. Ortega, D. Amoros, J. Garcia de La Torre, Biophys. J. 2011, 101, 892
898.
[34] J. Kohlbrecher, SASfit: A Program for fitting Simple Structural Models to
Small Angle Scattering Data, Villigen, Paul Scherrer Institut, 2008.
[35] D. E. Koppel, J. Chem. Phys. 1972, 57, 4814 4820.
[36] B. J. Berne, R. Pecora, Dynamic Light Scattering: With Applications to
Chemistry, Biology, and Physics, Dover, New York, 2000.
[37] S. Provencher, Comput. Phys. Commun. 1982, 27, 229 242.
[38] A. K. Rizos, D. A. Krambovitis, E. Spandidos, Int. J. Mol. Med. 2003, 12,
559 563.
[39] J. Horwitz, Proc. Natl. Acad. Sci. USA 1992, 89, 10449 10453.
[40] B. Raman, C. M. Rao, J. Biol. Chem. 1994, 269, 27264 27268.
[41] D. A. Haley, M. P. Bova, Q. L. Huang, H. S. Mchaourab, P. L. Stewart, J.
Mol. Biol. 2000, 298, 261 272.
[42] K. P. Das, J. M. Petrash, W. K. Surewicz, J. Biol. Chem. 1996, 271, 10449
10452.
[43] D. W. Hook, J. J. Harding, Int. J. Biol. Macromol. 1998, 22, 295 306.
[44] D. P. Claxton, P. Zou, H. S. Mchaourab, J. Mol. Biol. 2008, 375
, 1026
1039.
[45] J. W. Regini, H. Ecroyd, S. Meehan, K. Bremmell, M. J. Clarke, D. Lammie,
T. Wess, J. A. Carver, Mol. Vision 2010, 16, 2446 2456.
[46] G. B. Reddy, P. A. Kumar, M. S. Kumar, IUBMB Life 2006, 58, 632 641.
[47] T. Parasassi, M. De Spirito, G. Mei, R. Brunelli, G. Greco, L. Lenzi, G. Mau-
lucci, E. Nicolai, M. Papi, G. Arcovito, S. C. Tosatto, F. Ursini, FASEB J.
2008, 22, 2350 2356.
[48] D. Boyle, S. Gopalakrishnan, L. Takemoto, Biochem. Biophys. Res.
Commun. 1993, 192, 1147 1154.
[49] M. J. Clarke, J. B. Artero, M. Moulin, P. Callow, J. A. Carver, P. C. Griffiths,
M. Haertlein, J. J. Harding, K. M. Meek, P. Timmins, J. W. Regini, Biochim.
Biophys. Acta Gen. Subj. 2010, 1800, 392 397.
[50] M. De Spirito, R. Brunelli, G. Mei, F. R. Bertani, G. Ciasca, G. Greco, M.
Papi, G. Arcovito, F. Ursini, T. Parasassi, Biophys. J. 2006, 90, 4239 4247.
Received: July 8, 2013
Published online on November 12, 2013
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2362 2370 2370
CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
Page 9
  • Source
    • "SAXS data were collected at the BM29 beamline at the European Synchrotron Radiation Facility (ESRF) of Grenoble. Data were analysed as explained elsewhere [15, 18]. Briefly, for accurate subtraction of the scattering deriving from cell and solvent, buffer scattering was measured. "
    [Show abstract] [Hide abstract] ABSTRACT: High proteolytic degradation and poor absorption through epithelial barriers are major challenges to successful oral delivery of therapeutics. Nanoparticle platforms can enhance drug stability and extend the residence time in gastrointestinal (GI) tract. However, drug delivery systems are often inactivated in acidic environment of stomach or suffer poor absorption from intestinal cells due to the mucus layer. To overcome these issues we developed a drug delivery system constituted by a protein construct made by a Rotavirus capsid protein (VP6) and the small ubiquitin-like modifier SUMO. This chimeric construct allows specificity towards intestinal cells, the Rotavirus natural target, combined by an enhanced stability given by the eukaryotic protein transporter SUMO. Furthermore SUMO can act as a molecular switch that facilitates import/export of its ligand to the nucleus, the hypersensitive subcellular site target of many cell killing therapies. In this paper we show that SUMO-VP6 constructs self-assembly into stable nanocarriers. SUMO-VP6 nanocarriers display ideal features for drug delivery: a small size and high monodispersity, a high stability in different pH conditions and a high uptake in the nuclear and cytoplasmic compartment of intestinal cells. These features make SUMO-VP6 nanocarriers a promising novel system for oral delivery of poorly soluble drugs.
    Full-text · Article · Jan 2015 · Journal of Nanomaterials
  • [Show abstract] [Hide abstract] ABSTRACT: Provenance awareness adds a new dimension to the engineering of service-oriented systems, requiring them to be able to answer questions about the provenance of any data produced. This need is even more evident where atomic services are aggregated into added-value composite services to be delivered with certain non-functional characteristics. Prior work in the area of provenance for service-oriented systems has primarily focused on the collection and storage infrastructure required for answering provenance questions. In contrast, in this paper we study the structure of the data thus collected considering the service's infrastructure as a whole and how this affects provenance collection for answering different types of provenance questions. In particular, we define an extension of W3Cs PROV ontological model with concepts that can be used to express the provenance of how services were discovered, selected, aggregated and executed. We demonstrate the conceptual adequacy of our model by reasoning over provenance instances for a composite service scenario.
    No preview · Conference Paper · Apr 2014
  • [Show abstract] [Hide abstract] ABSTRACT: The structural properties of α-crystallin, the major protein of the eye lens of mammals, in aqueous solution are investigated by means of Small Angle X-ray and Dynamic Light Scattering. The research interest is devoted in particular to the effect of carnosine in protecting the protein under stress conditions, like temperature increase and presence of denaturant (guanidinium-HCl). The results suggest that carnosine interacts, through mechanisms involving hydrophobic interactions, with α-crystallin and avoids the structural changes in the quaternary structure induced by thermal and chemical stress. It is also shown that, if mediated by carnosine, the self-aggregation of α-crystallin induced by the denaturant at higher temperature can be controlled and even partially reversed. Therefore, carnosine is effective in preserving the structural integrity of the protein, suggesting the possibility of new strategies of intervention for preventing or treating pathologies related to protein aggregation, like cataract.
    No preview · Article · Oct 2014 · The Journal of Physical Chemistry B
Show more