C-terminal lysine truncation increases thermostability and
enhances chaperone-like function of porcine aB-crystallin
q
Jiahn-Haur Liao,
aJiahn-Shing Lee,
band Shyh-Horng Chiou
a,c,*a
Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
b
Department of Ophthalmology, Chang-Gung Memorial Hospital, Taipei, Taiwan
c
Laboratory of Crystallin Research, P.O. Box 23-106, Institute of Biological Chemistry, Academia, Nankang, Taipei 11529, Taiwan Received 7 August 2002
Abstract
The carboxyl-terminal segment of a-crystallin, a major lens protein of all vertebrates, has a short and flexible peptide extension of
about 20 amino acid residues that are very susceptible to proteolytic truncation and modifications under physiological conditions.
To investigate its role in crystallin aggregation and chaperone-like activity, we constructed a mutant of porcine aB-crystallin with
C-terminal lysine truncated end, which unexpectedly showed better chaperone-like function than wild-type aB-crystallin. From
circular dichroism (CD) spectra, we show that the mutant possesses similar secondary and tertiary structures to those of native
purified and recombinant aB-crystallins. Analytical ultracentrifugation revealed that the truncated mutant was smaller than
wild-type aB-crystallin in aggregation size and mass. The observed higher thermostability and anti-thermal aggregation propensity of the
truncated aB-crystallin mutant than wild-type aB-crystallin are in contrast to the prevailing notion that mutations at the C-terminal
lysines of aB-crystallin result in substantial loss of chaperone-like activity, despite the overall preservation of secondary structure.
The detailed characterization of the C-terminal deletion mutants may provide some deeper insight into the chaperoning mechanism
of the structurally related small heat-shock protein family.
Ó 2002 Elsevier Science (USA). All rights reserved.
Keywords: aB-crystallin; C-terminal truncation mutant; Chaperone activity; Thermostability; Circular dichroism; Small heat-shock proteins
a-Crystallin extracted from mammalian eye lenses
exists in solution as polydisperse aggregates ranging
from 600 to 1200 kDa with an average molecular mass
of approximately 800 kDa [1]. There are two a-crystallin
genes that encode two a-crystallin subunits known as
aA and aB, resulting in two polypeptides of about
20 kDa in size and 55–60% sequence similarity.
aA-crystallin is usually the more abundant subunit in the
lens, although the aA/aB ratio varies considerably
among species. They were previously regarded as
lens-specific proteins and of exclusively structural nature.
Ingolia and Craig first reported that heat-shock proteins
of Drosophila showed sequence similarity to mammalian
a-crystallin [2]. Later, it was demonstrated that
a-crys-tallin could function like heat-shock proteins by acting
as molecular chaperones in suppressing the aggregation
of other proteins in vitro [3]. aA-crystallin is highly
specialized for expression in the lens [4]; but
aB-crys-tallin is shown to be a functional small heat-shock
protein, which is ubiquitously present in various tissues
[5–7].
The small heat-shock proteins (sHSPs) form a
struc-turally divergent protein family with members present in
archaea, bacteria, and eukarya [8,9]. All members of the
sHSP family are characterized by the presence of a
ho-mologous sequence of about 80 residues, which has been
called the ‘‘a-crystallin domain’’ [2,10,11]. This domain
is preceded by an N-terminaldomain, which is highly
variable in size and sequence, followed by a short and
poorly conserved C-terminal extension. It was found
that C-terminaltruncated aA-crystallin by trypsin
di-gestion showed a decreased ability to protect proteins
from heat-induced aggregation using an in vitro assay
Biochemicaland BiophysicalResearch Communications 297 (2002) 309–316
www.academicpress.com
BBRC
q
Abbreviations: aB-crystallin, homoaggregate formed by association of aB crystallin subunits; sHSPs, small heat-shock proteins; CD, circular dichroism; ANS, 8-anilino-1-naphthalene sulfonate; PBS, phosphate-buffered saline.
*
Corresponding author. Fax: +886-2-26530014.
E-mail address:[email protected](S.-H. Chiou).
0006-291X/02/$ - see front matterÓ 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 1 8 5 - X
[12]. According to NMR analyses, the C-terminal 8 and
10 residues of aA- and aB-crystallin, respectively,
ap-pear to adopt a conformation as a solvent-exposed
random coil[13]. The presence of an exposed C-terminal
extension was in accord with the observation that the
last 20 or so residues of the a-crystallin are especially
liable to truncations and modifications [14–18]. A
mu-tant of aA-crystallin in which hydrophobicity was
in-troduced into the C-terminalextension was shown to
behave significantly less flexible and a corresponding
reduction in solubility, heat stability, and chaperone
activity when compared with the wild-type protein [19].
A mutant of mouse heat-shock protein (Hsp25) without
this extension was also constructed and shown to exhibit
a chaperone activity comparable to that of wild-type
Hsp25 in thermalaggregation assay using citrate
syn-thase as substrate, but does not stabilize a-lactalbumin
against precipitation following reduction with
dith-iothreitol [20]. Overall, previous structural studies
suggested that a highly flexible C-terminal extension in
mammalian sHSPs or the related aA or aB crystallins
appeared to be a prerequisite for full chaperone
activity.
We have previously cloned and expressed porcine
aB-crystallin [21], useful for site-specific mutagenesis to
derive the structure–function correlation of this
molec-ular chaperone. In this report, we have prepared
C-ter-minall
ysine
truncated
mutants
of
aB-crystallin,
examined and compared the structuraland functional
properties of these lysine-deleted mutants with wild-type
recombinant aB, and purified aB crystallin from native
lens by various biophysical methods. The results
ob-tained from these combined biophysicalapproaches
provide some insight into the structuralbasis of
C-ter-minal lysine residues in relation to the chaperone
activity of intact aB-crystallin.
Materials and methods
Isolation of lens crystallins. Porcine lenses were decapsulated and homogenized in a buffer containing 50 mM Tris–HCl, 0.1 M NaCl, 5 mM EDTA, 0.01% b-mercaptoethanol, and 0.02% sodium azide, pH 8.0. After centrifugation at 27,000g for 30 min, the supernatant was applied to a column packed with TSK HW-55(F) gel and eluted at 25 ml/h. Five well-resolved peaks were obtained and identified as HMa-, a-, bH-, bL-, and c-crystallins based on SDS–PAGE.
Construction of expression vector. The cDNA coding for porcine aB-crystallin was cloned and expressed as described previously [21]. The mutant cDNA fragments were made by polymerase chain reac-tion.
The same forward primer (50-GCCATATGGACATCGCCATC
CACCACC-30) was used for recombinant wild-type aB and
lysine-deleted mutant (T1, minus two lysine residues) crystallins.
The reverse primers used for wild-type aB and T1 were:
50-GCAAGCTTCTACTTCTTGGGGGCTGCAG-30 (WT-aB) and
50-GCAAGCTTCTAGGGGGCTGCAGTGACAGC-30 (T1),
respec-tively. The PCR products were double-digested with NdeI and HindIII and then ligated into Escherichia coli expression vector pET21a(+).
Expression and purification of porcine aB-crystallin and its mutants. Escherichia coli strain BL21(DE3) was transformed using above-mentioned expression constructs. The cells were incubated at 37°C untilthe culture reached an opticaldensity of 0.6 at 600 nm. Enhanced protein expression was assisted by addition of isopropyl-b-D -thioga-lactopyranoside (IPTG) to a final concentration of 1 mM. Four hours after incubation, cells were harvested, resuspended in lysis buffer (20 mM Tris–HCl, 25 mM NaCl, 1 mM EDTA, and 8 M urea, pH 8), and lysed by ultrasonication. After ultrasonication, the mixture was then centrifuged for 20 min at 20,000g to remove any remaining in-soluble debris. Soluble recombinant proteins were purified by a TSK HW-55 gel filtration column and followed by reverse-phase HPLC (C4). The purified recombinant aB-crystallin (WT-aB) and T1 mutant were then lyophilized.
Refoldingand reconstitution of aB-crystallin and its mutants. Native aB (N-aB), recombinant WT-aB, and T1 mutant crystallins were solubilized in 8 M urea individually and then loaded onto a gel filtra-tion column (TSK HW-55 gel). The separated protein fracfiltra-tions were then collected and concentrated. The protein concentrations were de-termined by absorbance measurements using extinction coefficients that are calculated from amino acid sequence data of each protein [22]. Circular dichroism spectra. Circular dichroism spectra were per-formed on a JASCO J-715 spectropolarimeter. Protein concentrations were 1:8 105M (far-UV region) and 3:6 105M (near-UV region)
in the buffer of 10 mM Na2HPO4, 2 mM KH2PO4, and 3 mM KCland
saturated with NaF, pH 7.4. The far-UV CD spectra were the mean of five accumulations with a 0.1 cm light path. The near-UV CD spectra were the mean of 10 accumulations with a 1 cm light path.
Fluorescence spectroscopy. The intrinsic fluorescence spectra were recorded with a Hitachi F4010 fluorescence spectrophotometer by setting the excitation wavelength at 295 nm, a light slit of 5 nm for both excitation and emission modes. The concentrations of native aB (N-aB), recombinant WT-aB, and T1 mutant crystallins was adjusted to 6.5 lM in the buffer of 10 mM NaHPO4, 2 mM KH2PO4, 3 mM KCl,
and 0.1 M NaCl, pH 7.4. Surface hydrophobicity of porcine native aB (N-aB), WT-aB, and T1 mutant crystallins were also analyzed using the fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS). For each sample, 10 ll ANS in methanolic stock solution (0.1 M) was ad-ded to 1 mlof 0.3 mg/mlprotein solution and incubated at indicated temperatures for 1 h, respectively. The extrinsic fluorescence spectra were then measured with a Hitachi F4010 fluorescence spectropho-tometer by setting the excitation wavelength at 395 nm, a light slit of 5 nm for both excitation and emission modes.
Analytical ultracentrifugation analysis. The molecular weight of these crystallins under various conditions was determined by a Beckman XL-A analytical ultracentrifuge (Beckman Instruments, Fullerton, CA) with an An60Ti rotor. Sedimentation velocity was performed at 25,000 rpm with standard double-sector aluminum centerpieces at 20°C. The UV absorption of the cells was scanned every 5 min for 2 h. The data were then analyzed with the software provided by the manu-facturer and the software SEDFIT [23]. Sedimentation equilibrium was performed at 20°C in a six-channelepon centerpiece with the centri-fugation set at 4800 rpm for 18 h. The data were also analyzed with a software provided by Beckman. All samples were visually checked for clarity after ultracentrifugation and no precipitation was observed. Assays of in vitro chaperone-like activity. The chaperone-like ac-tivities of porcine native aB (N-aB), recombinant WT-aB, and T1 mutant aB-crystallins were studied by two assays. Porcine bL-crys-tallin was used as substrate in the assay at 58°C. The finalconcen-tration of bL-crystallin was 6.8 lM in the buffer of PBS. The molar ratio was 1:1 (bL-crystallin: chaperone crystallin). In c-crystallin ag-gregation assay at 70°C, porcine c-crystallin was used as substrate in finalconcentrations of 5 and 2.5 lM using PBS buffer. The molar ratio was 1:1 and 1:2 (c-crystallin:chaperone crystallin) when 5 or 2.5 lM porcine c-crystallin was used, respectively.
Thermal stability of porcine aB-crystallin and its mutants. The as-sayed proteins were in a PBS buffer containing 10 mM Na2HPO4,
2 mM KH2PO4, 3 mM KCl, and 0.1 M NaCl, pH 7.4. Each protein
sample with an identical concentration of 36 lM was heated in a temperature range of 20–80°C, continuously measuring turbidity at OD 360 nm. The anti-thermalaggregation capabilities of porcine na-tive aB (N-aB), recombinant WT-aB, and T1 mutant crystallins were assayed at 70°C. All three crystallins in a concentration of 7.2 lM were dissolved in a buffer containing 10 mM Na2HPO4, 2 mM KH2PO4,
3 mM KCl, and 0.1 M NaCl, pH 7.4. For each protein, triplicate samples were incubated at 70°C for 10 min and the turbidities as measured by light scattering at OD 360 nm were measured before and after incubation.
Results and discussion
Recent literature abounds with reports characterizing
a variety of a-crystallin mutants and very often with
inconsistent and contradictory findings. Since we have
previously cloned and expressed porcine aB-crystallin
[21], it is very usefulto apply site-specific mutagenesis
for deriving the structure–function correlation of this
molecular chaperone. In this report, we have focused on
the role of two C-terminal lysine residues present in
aB-crystallin in relation to the chaperone activity of this
certified member of sHSPs family. The results obtained
on the C-terminall
ysine truncated mutants by using
various biophysicalapproaches may provide some
in-sight into the structuralbasis of C-terminallysine
resi-dues on the flexibility of C-terminal segment and their
effect on the chaperone activity of intact aB-crystallin in
general.
Expression, purification, and characterization of
molecu-lar masses
Escherichia coli strain BL21(DE3) was transformed
with expression constructs of aB crystallins. After
in-cubation, cells were harvested and resuspended in lysis
buffer and lysed by ultrasonication. Soluble
recombi-nant proteins were purified by TSK HW-55 gelfiltration
and followed by reverse-phase HPLC (C4). The purified
recombinant aB-crystallin and T1 mutant were then
lyophilized. The molecular masses of porcine native aB
(N-aB), recombinant WT-aB, and T1 mutant (minus
two lysine residues) crystallins were analyzed by
elec-trospray mass spectrometry and confirmed to possess
correct molecular masses of these subunits (data not
shown). Lyophilized native aB (N-aB), recombinant
WT-aB, and T1 mutant were dissolved in 8 M urea,
refolding in a TSK HW-55(F) gel filtration column.
These aB-crystallins and T1 mutant were thus refolded
after removalof urea. The purity of these proteins was
shown to be >95% by SDS–PAGE.
Circular dichroism spectropolarimetry
The far-UV CD spectra of native aB (N-aB),
re-combinant WT-aB, and T1 mutant crystallins were very
similar. Based on the CD spectra analyzed by the
pop-ular algorithm program [24], the secondary structure
estimation for these proteins showed mainly b-sheet
structure, which was consistent with the prevailing
evi-dence that a-crystallin consists mostly of b-sheet
sec-ondary structure [25]. The near-UV CD spectra of
native aB (N-aB), recombinant WT-aB, and T1 mutant
were also found to be similar (data not shown),
indi-cating that the microenvironments of bulky tryptophan
and tyrosine residues in these three crystallins are
probably kept in similar milieu.
Intrinsic tryptophan fluorescence spectra and surface
hydrophobicity
Intrinsic fluorescence provides information about the
local conformation of tryptophan residues inside
pro-teins. Tryptophan residues were excited selectively by
irradiation at 295 nm and fluorescence emission spectra
were then collected from 300 to 400 nm. The emission
maxima were located at 339–340 nm. All three
crystal-lins show similar intrinsic fluorescence spectra (Fig. 1A),
indicative of the similar microenvironments of
trypto-phan residues in three crystallins.
The fluors can be introduced into the molecule to be
studied either by chemical coupling or by simple
bind-ing. Such extrinsic protein fluorescence generated by the
use of such added molecules is sensitive for structural
comparison of homologous proteins. The extrinsic flour
used in our study is 8-anilino-1-naphthalene sulfonate
(ANS). It becomes fluorescent when bound to the
hy-drophobic area on the surface of various
macromole-cules. To investigate if there exists any surface
hydrophobicity difference among native aB (N-aB),
re-combinant WT-aB, and T1 mutant crystallins, we
pro-bed the surface hydrophobicity of these three crystallins
using ANS. The ANS fluorescence spectra (Fig. 1B) of
three proteins appear to be similar, which indicate that
native purified aB, wild-type aB, and T1 possess similar
surface hydrophobicity.
Analytical ultracentrifugation analysis
Further investigation on the quaternary structures of
native aB (N-aB), recombinant WT-aB, and T1 mutant
crystallins was performed by analytical ultracentrifuge.
Both sedimentation velocity and sedimentation
equi-librium have been used to characterize the particle sizes
of these three crystallins. Sedimentation velocity was
performed at 25,000 rpm with standard double-sector
aluminum centerpieces at 20
°C. Sedimentation
coeffi-cients (S or Svedberg values) were determined with the
second moment method of analysis software. The
re-sults of 15.8, 14.8, and 13.4 S for native aB (N-aB),
recombinant WT-aB, and T1, respectively, were
ob-tained (Fig. 2). Continuous size distribution analysis of
the sedimentation data was fitted by the software
SEDFIT program [23]. The fitted sedimentation
coeffi-cient distribution curve reveals a broad distribution of
crystallin species. The sedimentation coefficient
distri-bution of native aB (N-aB) and recombinant WT-aB
crystallins was between 12 and 18 S, while the
distri-bution of T1 mutant was between 10 and 16 S. All
re-sults from different analysis methods pointed to the fact
that the sedimentation coefficient obtained for T1 is
smaller than those of native aB and recombinant
WT-aB crystallins. Sedimentation equilibrium at a
centrifu-gation speed of 4800 rpm was performed at 20
°C for
18 h. The molecular weights of native aB, recombinant
WT-aB, and T1 are 490,000, 463,000, and 339,000,
respectively, in accord with results of sedimentation
velocity.
Comparison of the chaperone activity under thermal stress
The differences in the chaperone activity of native
aB (N-aB), recombinant WT-aB, and T1 mutant
crystallins were characterized in an in vitro
aggrega-tion assay using b-crystallin and c-crystallin as target
proteins at 58
°C and 70 °C, respectively. All three
proteins show similar chaperone-like activity when
porcine bL-crystallin was used as substrate at 58
°C for
chaperone activity assay in a molar ratio (chaperone/
substrate) of 1:1 (data not shown). However, it was
found that although both native and recombinant
aB-crystallins lost the protective activity, the C-terminal
truncated T1 mutant crystallin retained the protective
activity when c-crystallin was used as substrate at
70
°C in a molar ratio of 1:1 (Fig. 3A). Moreover,
Fig. 2. Sedimentation velocity experiment of native aB, recombinant WT-aB, and T1 mutant. Continuous size distributions of native aB-crystallin (solid line), wild-type aB-aB-crystallin (dash line), and T1 truncated mutant (dot line) are represented as curves of sedimentation coefficient distribution. The sedimentation coefficient distribution of native aB and wild-type aB was between 12 and 18 S, while the dis-tribution of T1 truncated mutant was between 10 and 16 S. All results from different analysis methods show that the sedimentation coefficient of T1 is smaller than those of native aB and recombinant WT-aB crystallins.
Fig. 1. Fluorescence spectral analysis of native aB, recombinant WT-aB, and T1 mutant crystallins. (A) Intrinsic fluorescence. Spectra were recorded for native aB crystallin (solid line), wild-type aB-crystallin (dash line), and T1 truncated mutant (dot line). Tryptophan residues were excited selectively by irradiation at 295 nm. Fluorescence emis-sion spectra were recorded between 300 and 400 nm. All three proteins show similar intrinsic fluorescence spectra with the same wavelength of emission maxima. (B) Comparison of ANS fluorescence emission spectra for native purified aB-crystallin (solid line), recombinant wild-type aB-crystallin (dash line), and T1 truncated mutant (dot line). The ANS fluorescence spectra of T1 truncated mutant and native aB are similar and slightly lower than those of recombinant wild-type aB-crystallin, which indicate that three crystallins possess similar surface hydrophobicity.
when we increased the molar ratio for the chaperoning
crystallin by decreasing the amount of c-crystallin,
neither native nor recombinant aB-crystallin regained
their protective activity (Fig. 3B). In contrast, T1
truncation mutant showed significantly higher
protec-tive activity.
Thermal stability of native purified, wild-type
aB-crystal-lin, and T1 mutant
We study the protein stability by incubating native
purified, recombinant wild-type aB-crystallin, and T1
Fig. 3. Comparison of chaperone activities for native aB, recombinant WT-aB, and T1 mutant against thermalaggregation of porcine c-crystallin at 70°C. Porcine c-crystallin was used as substrate in a molar ratio (chaperone/c-crystallin) of 1:1 (A) and 2:1 (B). The incu-bation mixture contained c-crystallin, in the absence (solid line) and presence of native crystallin (dash line), recombinant wild-type aB-crystallin (dot line), and T1 (line with square). Both assays show that T1 mutant protein has the protective activity for c-crystallin at 70°C. Note that native aB and recombinant WT-aB are very unstable at 70°C and turn turbid more quickly than control c-crystallin as shown in (B).
Fig. 4. Comparison of thermalstability for native aB, recombinant WT-aB, and T1 mutant. (A) Native purified aB-crystallin (solid line), recombinant wild-type aB-crystallin (dash line), and T1 (dot line) of identicalconcentration (36 lM in 0.5 ml) were analyzed for their thermal stability by heating the samples from 20 to 80°C. Turbidity changes for each crystallin solution were followed continuously by measuring OD (opticaldensity) at 360 nm. (B) The anti-thermalag-gregation capabilities of native aB, recombinant wild-type aB, and T1 truncated mutant crystallins were assayed at 70°C. Samples were in-cubated at 70°C for 10 min and the extent of light scattering (OD 360 nm) was measured before (black bars) and after (white bars) in-cubation. It clearly indicates that T1 lysine truncated mutant is stable at 70°C.
mutant using identicalprotein concentration in a
tem-perature range of 20–80
°C. The solutions of native
purified and wild-type aB-crystallins become turbid at a
temperature of 62
°C (transition temperature for
aB-crystallin aggregation) or higher, whereas the solution of
T1 remains clear, even after heating up to 70
°C (Fig.
4A). To further investigate the anti-thermalaggregation
capability of these proteins, we incubated these proteins
at 70
°C for 10 min and then measured the extent of light
scattering at 360 nm. For each protein, triplicate samples
were incubated at 70
°C and light scattering was
mea-sured before and after incubation (Fig. 4B). It is evident
that
light
scattering
of
native
and
recombinant
aB-crystallins increased sharply after incubation at
70
°C; however, T1 remained clear with very low light
scattering.
Conformational change of wild-type aB-crystallin and T1
mutant upon heating
To investigate the structuralchange of recombinant
wild-type aB-crystallin and T1 induced by heating, we
have employed circular dichroism to monitor the
con-formationalperturbation at different temperatures.
Es-timation of the secondary-structure composition of
these two crystallins based on CD at different
tempera-tures (Fig. 5) was carried out by SELCON3 algorithm
program using the expanded reference sets of denatured
proteins [24]. By increasing temperature from 20 to
60
°C, wild-type aB-crystallin showed an increase in the
proportion of unordered structure relative to b-sheet. In
contrast, T1 mutant appears to resist major structural
change even at 60–70
°C, attesting to its
structuralsta-bility at high temperature.
Conclusion
There is a considerable interest in the functional role
of aB-crystallin, especially after it was shown to be
ex-pressed in various non-lens tissues and is also found to
be overexpressed in some tissues of distinct pathological
states, such as retinoblastoma [26] and
neurodegenera-tive diseases [27,28]. It is well known that during aging
of the normallens, a-crystallins undergo extensive
post-translational modification, including especially
proteo-lytic cleavage from the C-terminus of the molecule [1].
C-terminall
ysine truncated aB-crystallin was also
re-ported to constitute between 10% and 90% of the total
intact aB-crystallin for human cataractous lenses [14]. In
this study, we focus on the comparison of the structure
and chaperone activity of native and recombinant
aB-crystallins, plus C-terminal lysine truncated mutant. It is
very intriguing to find that C-terminallysine truncated
mutant with higher thermostability actually performs
better as a molecular chaperone than the intact
aB-crystallin. Previously limited tryptic digestion on intact
a-crystallin showed that C-terminal truncated
aA-crys-tallin exhibited a decreased ability to protect proteins
from heat-induced aggregation using an in vitro assay
[12]. Moreover, mutations to the C-terminall
ysines
Fig. 5. The CD spectralchanges of recombinant wild-type aB and T1 truncated mutant by heating at different temperatures. (A) The CD spectra of wild-type aB-crystallin at 20°C (solid line with square), 40°C (solid line with circle), 50 °C (dot line), 55 °C (dash line), and 60°C (solid line). (B) The CD spectra of T1 at 20 °C (solid line with square), 40°C (solid line with circle), 50 °C (dot line), 60 °C (dash line), and 70°C (solid line). Secondary structural estimation based on CD spectra using SELCON3 algorithm program [24] revealed that by in-creasing temperature from 20 to 60°C, wild-type aB-crystallin showed an increase in the proportion of unordered structure relative to b-sheet. In contrast, T1 mutant appeared to resist major structuralchange even at 60–70°C, attesting to its structuralstability at high temperatures.
(Lys-174 and Lys-175) greatly diminished the
chaper-one-like activity of aB-crystallin [29]. Paradoxically,
substitution of Lys174–175 of aB-crystallin with Leu–
Leu significantly diminished chaperone activity;
how-ever, removal of the last five residues had little effect on
chaperone activity [30]. Therefore, both hydrophobic
and charge–charge interactions probably contribute to
the chaperone-like function of aB-crystallin.
In this study, we clearly demonstrate that T1 lysine
truncated mutant (minus Lys-174 and Lys-175) is more
stable than aB-crystallin at high temperature. Recently,
the crystalstructure of wheat sHSP 16.9 was sol
ved,
revealing the mode how a-crystallin domain and
flanking extensions can assemble into a dodecameric
double disk [31]. The ability of the C-terminal
exten-sion to build different assemblies stems from a hinge
between b9 and b10 strands that allow the angle
between the a-crystallin domain and the C-terminal
extension to vary by 30°, thus, providing some mobile
flexibility of C-terminal segment. Such a hinge
mecha-nism may contribute to the size polydispersity observed
in the assemblies of most sHSPs and a-crystallin
subunits. The elimination of two positive Lys–Lys
residues from the C-terminalsegment may decrease
charge–charge interaction of C-terminalcoilwith other
parts of aB-crystallin, thus, increasing the adaptability
of C-terminalextension to bind the unfolded substrate
polypeptides and promoting chaperone activity. We
have recently demonstrated that both surface
hydro-phobicity and structural stability play some roles in the
molecular mechanism underlying the chaperone-like
activity of a-crystallin and its subunit aggregates [32].
As a member of small heat-shock proteins,
aB-crystallin may be a better chaperone than aA-aB-crystallin
under most thermalstress. The creation of C-terminal
truncated mutants with high thermostability and
anti-thermalaggregation propensity may prove to be a
better molecular chaperone suitable for detailed
func-tionalcharacterization.
Acknowledgments
This work was supported in part by Academia and the National Science Council(NSC Grants 87-2311-B-002-068, 88-2311-B-002-061, and 89-2311-B-001-190 to S.-H. Chiou), Taipei, Taiwan. This report will be submitted as part of a dissertation by J.-H. Liao to National Taiwan University in partial fulfillment of the degree of Doctor of Philosophy. We thank Professor Gu-Gang Chang at the Department of Biochemistry, NationalDefense MedicalCenter, Taiwan, for assisting sedimentation velocity and equilibrium analyses.
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