THE JOURNAL OF BIOLOGICAL CHEMISTRY
47094 –47099, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Phe71 Is Essential for Chaperone-like Function in ␣A-crystallin*
Received for publication, August 13, 2001, and in revised form, September 21, 2001
Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M107737200
Puttur Santhoshkumar‡ and K. Krishna Sharma‡§¶
From the Departments of ‡Ophthalmology and §Biochemistry, University of Missouri, Columbia, Missouri 65212
Experiments with mini-␣A-crystallin (KFVIFLD-
VKHFSPEDLTVK) showed that Phe71 in ␣A-crystallin
could be essential for the chaperone-like action of the
protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and
Quinn, P. T. (2000) J. Biol. Chem. 275, 3767–3771). In the
present study we replaced Phe71 in rat ␣A-crystallin
with Gly by site-directed mutagenesis and then com-
pared the structural and functional properties of the
mutant protein with the wild-type protein. There were
no differences in molecular size or intrinsic tryptophan
fluorescence between the proteins. However, 1,1ⴕ-bi(4-
anilino)naphthalene-5,5ⴕ-disulfonic acid interaction in-
dicated a higher hydrophobicity for the mutant protein.
Both wild-type and mutant proteins displayed similar
secondary structure during far UV CD experiments.
Near UV CD signal showed a slight difference in the
tertiary structure around the 285–295 region for the two
proteins. The mutant protein was totally inactive in sup-
pressing the aggregation of reduced insulin, heat-dena-
tured citrate synthase, and alcohol dehydrogenase.
However, a marginal suppression of ␤L-crystallin aggre-
gation was observed when mutant ␣A-crystallin was in-
cluded. These results suggest that Phe71 contributes to
the chaperone-like action of ␣A-crystallin. Therefore we
conclude that the 70 – 88-region in ␣A-crystallin, identi-
fied by us earlier, is the functional chaperone site in
␣A-crystallin.
␣-Crystallins are major refractive proteins in the vertebrate
eye lens. When isolated from the lens they exist as polydisperse
aggregates having an average molecular mass of ⬇800 kDa (1,
2). ␣-Crystallin is composed of two subunits, ␣A and ␣B, which
have considerable sequence homology between them and with
other heat shock proteins (3, 4). Recently ␣-crystallin subunits
were also reported to be present in nonlenticular tissues like
heart, brain, and kidney (5– 8). The significance of their pres-
ence in nonlenticular tissues is not clear. However, the in-
creased expression of ␣B-crystallin observed in a variety of
neurological disorders has drawn significant medical attention
(9 –11). Like other small heat shock proteins, ␣-crystallin can
sequester certain unfolding proteins in vitro, by preventing
their aggregation and insolubilization (12–15). Both subunits
of ␣-crystallin show chaperone-like activity to different extents
(16 –18). Complex formation with ␤L- and ␥-crystallin and de-
creased chaperone-like function during aging has indicated the
importance of ␣-crystallin in maintaining the transparency of
* This work is supported in part by National Institutes of Health
Grant EY11981 and a grant-in-aid from Research to Prevent Blindness.
The costs of publication of this article were defrayed in part by the
payment of page charges. This 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: Mason Eye Inst.,
Dept. of Ophthalmology, 1 Hospital Dr., University of Missouri, Colum-
bia, MO 65212. E-mail: Sharmak@health.missouri.edu.
47094
Vol. 276, No. 50, Issue of December 14, pp.
Printed in U.S.A.
the lens (2, 19 –21). During chaperone-like action, hydrophobic
surfaces in ␣-crystallin interact with specific sites in non-na-
tive target proteins (22–24). Earlier we were able to map the
site in ␣A- and ␣B-crystallin responsible for chaperone-like
action using photoactive cross-linkers and hydrophobic probes
(25–27). Our studies with bis-ANS1 and the hydrophobic pro-
tein mellitin have shown that there is an overlapping of chap-
erone site and hydrophobic site in ␣A-crystallin. Further, using
a synthetic peptide (mini-␣A-crystallin), we were able to dem-
onstrate the importance of sequence 70 – 88 in the chaperone-
like action of ␣A-crystallin (28). The experiments with trun-
Downloaded from www.jbc.org by guest, on October 28, 2010
cated forms of mini-␣A-crystallin had suggested that Phe71 in
␣A-crystallin might be critical for chaperone-like function. In
the present study, we did a site-directed mutagenesis of Phe71
to Gly in ␣A-crystallin and compared the structural and func-
tional properties of this mutant protein with the wild-type
protein.
Recombinant ␣A- and ␣B-crystallins show similar structural
and functional properties to crystallins isolated from lens tis-
sues and are widely used in the characterization of the protein.
Site-directed mutations of recombinant protein provide an ex-
cellent means of studying the role of constituent amino acids in
the functional properties of the protein. Earlier, several site-
directed mutations were conducted on ␣-crystallin either to
identify the region responsible for chaperone-like function or to
explain the role of ␣-crystallin in hereditary cataracts and
certain other diseases (29 –38). The majority of these studies
report either no change in chaperone-like function or a partial
loss of this function. We report here, for the first time, a
complete loss in the functional property of a mutant ␣A-crys-
tallin at and slightly above physiological temperatures. The
results also indicate the presence of additional sites in ␣-crys-
tallin that become available at elevated temperatures. We con-
clude here that the region identified by us earlier as chaperone
site contributes to the chaperone-like activity of ␣A-crystallin.
EXPERIMENTAL PROCEDURES
Preparation of the Mutant Clone—Rat ␣A-crystallin cDNA cloned in
pET21b was kindly donated by Dr. Suraj Bhat (UCLA). ␣AF71G mu-
tant was constructed using a QuikChange site-directed mutagenesis kit
(Stratagene). The following set of primers were used: 5⬘-CTGACCGG-
GACAAGGGTGTCATCTTCTTGG-3⬘ and 5⬘-CCAAGAAGATGACAC-
CCTTGTCCCGGTCAG-3⬘. The mutation was confirmed by automated
DNA sequencing.
Expression and Purification of Wild-type and Mutant ␣A-crystallin—
The proteins were expressed in Escherichia coli BL21(DE3) cells (No-
vagen) as described by Horwitz et al. (39). The proteins were isolated
from the cell pellet using Bugbuster protein extract reagent (Novagen).
In brief, 1 g of cells was suspended in 5 ml of reagent at room temper-
ature and vortexed gently. Protease inhibitor mixture set III (Novagen)
was then added. The cell suspension was treated with 1 ␮l (25 units) of
benzonase/ml of Bugbuster reagent and incubated at room temperature
on a shaking platform for 30 min. The extract was centrifuged at
1
The abbreviations used are: bis-ANS, 1,1⬘-bi(4-anilino)naphthalene-
5,5⬘-disulfonic acid; CS, citrate synthase; ADH, alcohol dehydrogenase;
HPLC, high pressure liquid chromatography.
This paper is available on line at http://www.jbc.org
 F71G Mutant of ␣A-crystallin 47095
17,000 ⫻ g for 2 h, and the supernatant was filtered through a 0.2-␮m
filter. The filtrate was loaded onto a Bio-Rad High Q anion exchange
column and eluted using a linear gradient of NaCl (0 –1 M) in 20 mM
Tris-HCl (pH 8) at a flow rate of 2 ml/min. The fractions containing the
recombinant crystallin (determined by SDS-PAGE) were pooled and
concentrated. The concentrated protein was purified further on a C18
reverse phase HPLC using a water-acetonitrile gradient containing
0.1% trifluoroacetic acid. The peaks corresponding to wild-type and
mutant ␣A-crystallin were pooled, dried on a Speedvac, resuspended in
6 M urea, and dialyzed extensively against 0.05 M phosphate (PO4)
buffer containing 0.15 M NaCl, with several changes, for a period of 2
days. The purity of the proteins was checked by SDS-PAGE, and the
mass was determined by mass spectrometry.
Molecular Size Determination—Size determination was carried out
using a Amersham Biosciences Hiload 16/60 Superdex 200 gel filtration
column equilibrated with 0.05 M PO4 buffer containing 0.15 M NaCl (pH
7.4). The mass was calculated from the calibration curve generated by FIG. 1. SDS-PAGE analysis of recombinant protein purifica-
using Sigma molecular weight marker standards. tion. Lane 1, crude cell extract; lane 2, recombinant protein after ion
Tryptophan Fluorescence Measurements—The intrinsic fluorescence exchange; lane 3, recombinant protein after reverse phase; lane 4,
spectra of the wild-type and mutant ␣A-crystallin were recorded using molecular mass markers.
a Jasco spectrofluorometer FP-750. Protein samples of 200 ␮g/ml in
0.05 M PO4 buffer containing 0.15 M NaCl were used. The excitation was
set to 295 nm, and the emission was recorded between 300 and 400 nm.
bis-ANS Fluorescence Measurement—To 100 ␮g of wild-type and
mutant protein taken in 0.05 M PO4 containing 0.15 M NaCl (pH 7.4)
was added 20 ␮l of 10 mM bis-ANS dissolved in ethanol. The sample was

Downloaded from www.jbc.org by guest, on October 28, 2010

excited at 365 nm, and the fluorescence spectrum was measured be-
tween 400 and 600 nm using a Jasco spectrofluorometer.
Circular Dichroism Studies—Protein secondary and tertiary struc-
tural changes were investigated by far and near UV CD measurements
using an AVIV circular dichroism spectrometer. The concentration of
the proteins used was 1.5 and 0.35 mg/ml for near and far UV CD,
respectively. The reported CD spectra are the averages of four scans.
Chaperone-like Activity—The ability of the wild-type and mutant
proteins to prevent protein aggregation was determined using several
substrates. The extent of aggregation was measured by monitoring the
light scattering at 360 nm in a Shimadzu spectrophotometer.
Insulin Aggregation Assay—The aggregation of insulin (0.4 mg/ml)
(Sigma) in 0.05 M PO4 buffer containing 0.15 M NaCl (pH 7.2) was
initialized by the addition of 25 ␮l of 1 M dithiothreitol in the presence
of wild-type and mutant proteins. The aggregation was monitored at
room temperature.
CS Aggregation Assay—CS (75 ␮g) (Roche Molecular Biochemicals)
in 1 ml of 40 mM HEPES-NaOH buffer (pH 7.4) was heated at 43 °C for
1 h in the presence of various amounts of mutant and wild-type pro-
teins. The light scattering was measured as described above.
ADH Aggregation Assay—ADH (250 ␮g) (Sigma) was heated at 45 °C
in the presence of various amounts of mutant and wild-type ␣A-crys-
tallin in 0.05 M PO4 buffer containing 0.15 M NaCl (pH 7.4). The
scattering of light was measured up to 100 min.
␤L-Crystallin Aggregation Assay—Purified bovine ␤L-crystallin (250
␮g) in 0.05 M PO4 buffer containing 0.15 M NaCl (pH 7.4) was heated at
55 °C in the presence of different amounts of recombinant proteins. The FIG. 2. Gel permeation profiles of refolded wild-type and mu-
aggregation was monitored up to 1 h. tant protein on a Superdex 200 column.

RESULTS also observed a similar mass for reconstituted homopolymers of

Characterization of Recombinant ␣A-crystallin—In the pres-
ent study, the recombinant proteins were purified by a combi-
nation of ion exchange and reverse phase HPLC. The purified
proteins were dissolved in urea and refolded by extensive dial-
ysis. The proteins thus obtained were highly pure (Fig. 1). The
ES mass spectrometry analysis revealed molecular masses of
19,799 and 19,709 daltons, which would be expected for the
wild-type and mutant ␣A-crystallin respectively. Like the
␣-crystallin subunits isolated from eye lens, recombinant pro-
teins exist in oligomeric form. For analysis of the molecular
mass of the homoaggregates, the purified recombinant proteins
were chromatographed on a Superdex-200 column. Both wild-
type and ␣AF71G mutant proteins showed similar elution pro-
files, corresponding to an oligomeric mass of 7.1 ⫻ 105 daltons
(Fig. 2). This is slightly higher than the earlier published
values for rat ␣A-crystallin (37). The discrepancy can be attrib-
uted to the different buffer conditions used in analysis, because
it is known that the mass of the purified protein varies depend-
ing on the buffer condition (40, 41). During these studies we
bovine lens ␣A-crystallin.
The structural differences between the wild-type and mutant
proteins were analyzed by spectroscopic methods. Tryptophans
of the protein have a fixed solvent accessibility, and any change
in their environment leads to an altered fluorescence emission
pattern and intensity. Our results show no change in the tryp-
tophan region of the recombinant proteins, as evidenced by the
similar fluorescence emission maximum (340 nm) and intensity
(Fig. 3). Unfolding of proteins increases the exposure of hydro-
phobic surfaces that can be probed with bis-ANS fluorescence
(26). We see an increase in bis-ANS binding to the ␣AF71G
mutant (Fig. 4), indicating an increased hydrophobicity com-
pared with wild-type protein.
The secondary and tertiary structures of wild-type and mu-
tant ␣A-crystallin were determined by far and near UV CD
spectral analysis. The far UV profile showed a characteristic
␤-sheet conformation with a slight increase in the negative
intensity of the mutant protein (Fig. 5). Both proteins showed
similar amounts of ␣-helix, ␤-sheet, and random coil (42). Near
47096 F71G Mutant of ␣A-crystallin
FIG. 3 Intrinsic fluorescence intensity of purified wild-type
and mutant ␣A-crystallin. Protein samples (200 ␮g) in phosphate
buffer were excited at 295 nm.
FIG. 4. Interaction of bis-ANS with mutant and wild-type
␣A-crystallin.
UV CD spectra showed a slight increase in the negative inten-
sity of the mutant protein (Fig. 6). Although significant por-
tions of the near UV spectra for the two proteins were similar,
only minor changes were seen in the 285–295 nm region of the
spectra, suggesting some differences in the tyrosine and/or
tryptophan microenvironments of the mutant protein com-
pared with the wild-type ␣A-crystallin. Surprisingly enough,
there was no alteration in the signal caused by phenylalanine
in the 250 –270-nm region. In summary, the data in Fig. 6 do
not suggest a significant difference in the tertiary structure
between wild-type and mutant ␣A-crystallin.
The Chaperone-like Activity of F71G ␣A-crystallin—The con-
sequence of mutation on recombinant crystallin chaperone-like
activity was determined under different conditions. Reduction
Downloaded from www.jbc.org by guest, on October 28, 2010
FIG. 5 Far UV CD spectra of recombinant proteins.
FIG. 6. Near UV CD spectra of recombinant proteins.
of insulin results in the separation of the subunits and precip-
itation of B chain that can be followed by measurement of light
scattering. The presence of ␣-crystallin subunits in the assay
prevents the aggregation of insulin B chain, and the solution
remains clear. Fig. 7 shows the dithiothreitol-induced aggrega-
tion kinetics of insulin in the presence of both wild-type and
mutant ␣A-crystallins. The wild-type protein showed suppres-
sion of insulin B chain aggregation that increased with the
concentration of the protein in the assay tube. However, the
mutant ␣A-crystallin completely failed to prevent the forma-
tion of light-scattering aggregates. In fact, a marginal increase
in light scattering was observed in some assays. Higher con-
centrations of mutant protein had no effect on the aggregation
of polypeptide. The chaperone-like activity of the recombinant
proteins was also investigated at different temperatures. Fig. 8
 F71G Mutant of ␣A-crystallin
FIG. 7 Reduction of insulin (0.4 mg) by dithiothreitol in the
presence of wild-type and mutant ␣A-crystallin. A, insulin alone;
B–D, insulin with 0.1, 0.2, and 0.3 mg of mutant ␣A-crystallin, respec-
tively; E and F, insulin with 0.05 and 0.1 mg of wild-type ␣A-crystallin,
respectively.
FIG. 8. Aggregation of CS at 43 °C in the presence of wild-type
and mutant ␣A-crystallin. A, CS (75 ␮g); B, CS ⫹ mutant (50 ␮g); C,
CS ⫹ mutant (100 ␮g); D, CS ⫹ wild type (25 ␮g); E, CS ⫹ wild type (50
␮g).
shows the thermal aggregation of CS in the presence of wild-
type and mutant proteins. Although the wild-type protein (50
␮g) completely suppressed the aggregation of CS (75 ␮g), the
mutant protein, as with insulin, failed to prevent the aggrega-
tion of denaturing CS. We also analyzed the ability of recom-
binant proteins to suppress the aggregation of ADH at 45 °C.
The wild-type ␣A-crystallin showed increased suppression of
denaturing protein aggregation with increasing concentration
(Fig. 9A). Although the mutant protein appeared to suppress
the aggregation of ADH at initial time points, the aggregation
47097
Downloaded from www.jbc.org by guest, on October 28, 2010
FIG. 9. Thermal aggregation of ADH (250 ␮g) in the presence of
various amounts of wild-type and mutant ␣A-crystallin at 45 °C.
A, in the presence of wild type; B, in the presence of mutant
␣A-crystallin.
at 80 min was comparable with ADH by itself (Fig. 9B). In-
creasing the concentration of mutant protein had no effect on
the aggregation of ADH. We also compared the abilities of
mutant and wild-type ␣A-crystallin to prevent the heat-in-
duced aggregation of ␤L-crystallin at 55 °C (Fig. 10). Unlike
other substrates, mutant ␣A-crystallin showed a significant
protection of ␤L-crystallin with increasing concentration. How-
ever, compared with the wild-type ␣A-crystallin, the mutant
␣A-crystallin was 6 –10-fold less effective in suppressing
␤L-aggregation.
DISCUSSION
␣A-crystallin subunit has been categorized into three do-
mains: an N-terminal domain containing residues 1– 66, a C-
terminal or ␣-crystallin domain (central core) comprising resi-
dues 64 –105, and an extended C-terminal including residues
106 –173 (43– 45). Most of the mutational studies on ␣A-crys-
tallin were conducted either on the N-terminal domain or the
C-terminal extension. Derham and Harding (46) have reviewed
the mutations conducted by different laboratories on ␣-crystal-
lin and have recently reanalyzed the chaperone-like activity of
several mutants (35). In the present study, we produced an
␣A-crystallin mutant by substituting Phe71 in the core region
with a neutral amino acid Gly. This residue is highly conserved
in ␣A-crystallin and is located in the region identified as the
chaperone site of ␣A-crystallin (28). Biophysical characteriza-
tion of the recombinant protein revealed no change in the
oligomer size or tryptophan fluorescence. Because the ␣-crys-
tallin molecule has only one tryptophan at position 9, the
intrinsic tryptophan fluorescence data may be of limited value
47098 F71G Mutant of ␣A-crystallin
FIG. 10. Aggregation of ␤L-crystallin (250 ␮g) in the presence of
recombinant proteins. A, ␤L-crystallin; B, ␤L-crystallin ⫹ mutant ␣A
(100 ␮g); C, ␤L-crystallin ⫹ mutant ␣A (200 ␮g); D, ␤L-crystallin ⫹
mutant ␣A (300 ␮g); E, ␤L-crystallin ⫹ wild type ␣A (50 ␮g); F, ␤L-
crystallin ⫹ wild type ␣A (100 ␮g).
to describe the structural changes in the central core or C-
terminal domain as a consequence of the mutation. However, it
will be a valuable tool for analyzing the stability of the N-
terminal domain of the mutant protein. When the mutant
protein was heated up to 60 °C, we did not observe any aggre-
gation or shift in tryptophan fluorescence emission wavelength
or intensity (data not shown), suggesting that the heat stability
of the protein was not affected by the mutation.
It has been hypothesized that hydrophobic sites in ␣-crystal-
lin are responsible for chaperone-like activity (22, 23). How-
ever, this is not free of controversy (29, 47). In the present
study we see a complete loss in the chaperone-like function of
mutant ␣A-crystallin at and slightly above physiological tem-
peratures despite an increase in hydrophobicity. Smulders et
al. (29) observed an increase in the chaperone-like activity of an
␣AF74N mutant with a slight decrease in ANS binding. They
concluded that there is no correlation between surface hydro-
phobicity and chaperone-like activity. Experiments with super-
␣A-crystallin have indicated that the disappearance of chaper-
one-like activity may be independent of hydrophobicity (47).
Further, Reddy et al. (18) have shown recently that hydropho-
bicity is not the sole determinant of chaperone-like activity in
␣-crystallin. Recently, the studies with mini-␣A-crystallin
showed that both hydrophobicity and ␤-sheet conformation of
the functional element are essential for chaperone-like activity
(48). Although we see increased exposure of hydrophobic sur-
faces in the mutant, it is quite unlikely that all exposed hydro-
phobic patches would be involved in suppressing the substrate
protein aggregation. We, as well as others, have observed bis-
ANS binding to residues other than those necessary for chap-
erone activity (27, 49). Taking these observations together, one
can conclude that although hydrophobicity is important, the
extent of hydrophobicity does not reflect the chaperone-like
activity of the protein.
The ␣AF71G mutant has similar secondary structure to that
of the wild type. However, the tertiary structure shows some
minor changes around the 285–295-nm region. The signal in
this region is produced by tyrosine or tryptophan residues.
Because we did not observe any change in the tryptophan
fluorescence intensity, it is possible that the alteration is in the
tyrosine region. Interestingly, two tyrosine residues in ␣A-
crystallin are found near the bis-ANS-binding region 50 –54
(27). This may explain the increased bis-ANS binding of the
mutant protein. However, it is unlikely that such a minor
difference in the near UV CD signal would completely abolish
the chaperone-like activity of the molecule. The ␣AR116C mu-
tant, with structural alterations at many levels, showed only a
25% decrease in chaperone-like activity (37, 38). Further, it has
been shown that ␣-crystallin could preserve its chaperone func-
tion despite some irreversible structural changes (50).
We have measured the chaperone-like function of the
␣AF71G mutant under different conditions and observed a
complete loss in the activity of the mutant up to 45 °C. How-
ever, at elevated temperatures the mutant showed some sup-
pression of ␤L-crystallin aggregation. It has been shown that
␣-crystallin undergoes a structural transition around 55 °C,
resulting in the exposure of more hydrophobic patches (23, 24,
26). Our study on the stabilization of restriction enzyme (51) as
well as studies conducted by others (35, 52) indicates the pres-
ence of multiple sites in ␣-crystallin for chaperone function.
Based on the experiments with mini-␣A-crystallin (28) and the
Downloaded from www.jbc.org by guest, on October 28, 2010
complete loss of chaperone-like function of the mutant protein
at physiological temperatures in this study, we conclude that
the region identified by us earlier (residues 71– 88) contributes
to the chaperone-like function.
Plater et al. (30) reported that the F27R mutation in the
N-terminal domain of ␣B-crystallin completely abolishes its
chaperone-like activity at higher temperatures, which led them
to conclude that this conserved residue is vital for chaperone
function. Their report is controversial because later studies
have shown that the mutant F27R is fully active (34, 35).
Earlier, work showed that proteins resulting from mutation of
V72N and F74N in the core region of ␣A-crystallin had normal
activity (29). However, their conclusion was based on a single
assay conducted at 58 °C. We also found some activity of the
mutant with ␤L-crystallin around this temperature. Also, un-
like Phe71, the Val72 and Phe74 residues show variations in
different vertebrate lens species. The conserved Phe71 residue
appears to be important for suppressing the aggregation of
proteins. Other factors like charge, hydrophobicity, and struc-
tural integrity may influence the functional property to differ-
ent extents. Recently, Kumar and Rao (53) produced a chimeric
␣-crystallin by swapping the domains of ␣A- and ␣B-crystallin
and tested its effect on the chaperone-like activity. Interest-
ingly, the ␣ANBC chimeric protein which contained residues
1–79 of ␣A-crystallin, including a part of the functional site in
␣A-crystallin, was completely inactive in suppressing the ag-
gregation of insulin. However, the ␣BNAC chimeric protein
containing the complete ADH binding sequence (25) and a part
of the functional site in ␣A-crystallin had enhanced chaperone-
like activity. This suggests that other residues in the chaperone
site of ␣A-crystallin are also important in suppressing the
aggregation of proteins. Therefore it would be interesting to
study the role of other conserved residues on chaperone-like
action.
Acknowledgments—We are grateful to Dr. P. D. Prasad for the help
in site-directed mutagenesis and Jelena Kocergin in the expression and
purification of recombinant proteins.
REFERENCES
1. Harding, J. J., and Crabbe, M. J. (1984) in The Eye, pp. 207– 492, Academic
Press, New York
2. Groenen, P. J., Merck, K. B., de Jong, W. W., and Bloemendal, H. (1994) Eur.
J. Biochem. 225, 1–19
3. Ingolia, T. D., and Craig, E. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,
525–529
 F71G Mutant of ␣A-crystallin
4. Merck, K. B., Groenen, P. J., Voorter, C. E., de Haard-Hoekman, W. A.,
Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. 268,
1046 –1052
5. Bhat, S. P., and Nagineni, C. N. (1989) Biochem. Biophys. Res. Commun. 158,
319 –325
6. Dubin, R. A., Wawrousek, E. F., and Piatigorsky, J. (1989) Mol. Cell. Biol. 9,
1083–1091
7. Nagineni, C. N., and Bhat, S. P. (1989) FEBS Lett. 249, 89 –94
8. Kato, K., Shinohara, H., Kurobe, N., Inaguma, Y., Shimizu, K., and Ohshima,
K. (1991) Biochim. Biophys. Acta 1074, 201–208
9. van Noort, J. M., van Sechel, A. C., Bajramovic, J. J., el Ouagmiri, M., Polman,
C. H., Lassmann, H., and Ravid, R. (1995) Nature 375, 798 – 801
10. Agius, M. A., Kirvan, C. A., Schafer, A. L., Gudipati, E., and Zhu, S. (1999) Acta
Neurol. Scand. 100, 139 –147
11. Celet, B., Akman-Demir, G., Serdaroglu, P., Yentur, S. P., Tasci, B., van Noort,
J. M., Eraksoy, M., and Saruhan-Direskeneli, G. (2000) J. Neurol. 247,
935–939
12. Horwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10449 –10453
13. Wang, K., and Spector, A. (1994) J. Biol. Chem. 269, 13601–13608
14. Gopalakrishnan, S., Boyle, D., and Takemoto, L. (1994) Invest. Ophthalmol.
Vis. Sci. 35, 382–387
15. Wang, K., and Spector, A. (1995) Invest. Ophthalmol. Vis. Sci. 36, 311–321
16. van Boekel, M. A., de Lange, F., de Grip, W. J., and de Jong, W. W. (1999)
Biochim. Biophys. Acta 1434, 114 –123
17. Datta, S. A., and Rao, C. M. (1999) J. Biol. Chem. 274, 34773–34778
18. Reddy, G. B., Das, K. P., Petrash, J. M., and Surewicz, W. K. (2000) J. Biol.
Chem. 275, 4565– 4570
19. Takemoto, L., and Boyle, D. (1994) Arch. Biochem. Biophys. 315, 133–136
20. Rao, P. V., Huang, Q. L., Horwitz, J., and Zigler, J. S., Jr. (1995) Biochim.
Biophys. Acta 1245, 439 – 447
21. Carver, J. A., Nicholls, K. A., Aquilina, J. A., and Truscott, R. J. (1996) Exp.
Eye Res. 63, 639 – 647
22. Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264 –27268
23. Das, K. P., and Surewicz, W. K. (1995) FEBS Lett. 369, 321–325
24. Raman, B., and Rao, C. M. (1997) J. Biol. Chem. 272, 23559 –23564
25. Sharma, K. K., Kaur, H., and Kester, K. (1997) Biochem. Biophys. Res. Com-
mun. 239, 217–222
26. Sharma, K. K., Kaur, H., Kumar, G. S., and Kester, K. (1998) J. Biol. Chem.
273, 8965– 8970
27. Sharma, K. K., Kumar, G. S., Murphy, A. S., and Kester, K. (1998) J. Biol.
Chem. 273, 15474 –15478
28. Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol.
Chem. 275, 3767–3771
29. Smulders, R. H., Merck, K. B., Aendekerk, J., Horwitz, J., Takemoto, L.,
47099
Slingsby, C., Bloemendal, H., and De Jong, W. W. (1995) Eur. J. Biochem.
232, 834 – 838
30. Plater, M. L., Goode, D., and Crabbe, M. J. (1996) J. Biol. Chem. 271,
28558 –28566
31. Hepburne-Scott, H. W., and Crabbe, M. J. (1999) Mol. Vis. 5, 15–20
32. Muchowski, P. J., Wu, G. J., Liang, J. J., Adman, E. T., and Clark, J. I. (1999)
J. Mol. Biol. 289, 397– 411
33. Kumar, L. V., Ramakrishna, T., and Rao, C. M. (1999) J. Biol. Chem. 274,
24137–24141
34. Horwitz, J., Bova, M., Huang, Q. L., Ding, L., Yaron, O., and Lowman, S.
(1998) Int. J. Biol. Macromol. 22, 263–269
35. Derham, B. K., van Boekel, M. A., Muchowski, P. J., Clark, J. I., Horwitz, J.,
Hepburne-Scott, H. W., de Jong, W. W., Crabbe, M. J., and Harding, J. J.
(2001) Eur. J. Biochem. 268, 713–721
36. Bova, M. P., Yaron, O., Huang, Q., Ding, L., Haley, D. A., Stewart, P. L., and
Horwitz, J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6137– 6142
37. Shroff, N. P., Cherian-Shaw, M., Bera, S., and Abraham, E. C. (2000) Biochem-
istry 39, 1420 –1426
38. Cobb, B. A., and Petrash, J. M. (2000) Biochemistry 39, 15791–15798
39. Horwitz, J., Huang, Q. L., Ding, L., and Bova, M. P. (1998) Methods Enzymol.
290, 365–383
40. Augusteyn, R. C., Parkhill, E. M., and Stevens, A. (1992) Exp. Eye Res. 54,
219 –228
41. Siezen, R. J., Bindels, J. G., and Hoenders, H. J. (1980) Eur. J. Biochem. 111,
435– 444
42. Andrade, M. A., Chacon, P., Merelo, J. J., and Moran, F. (1993) Protein Eng. 6,
383–390
43. Caspers, G. J., Leunissen, J. A., and de Jong, W. W. (1995) J. Mol. Evol. 40,
238 –248
44. de Jong, W. W., Caspers, G. J., and Leunissen, J. A. (1998) Int. J. Biol.
Macromol. 22, 151–162
Downloaded from www.jbc.org by guest, on October 28, 2010
45. de Jong, W. W., Leunissen, J. A., and Voorter, C. E. (1993) Mol. Biol. Evol. 10,
103–126
46. Derham, B. K., and Harding, J. J. (1999) Prog. Retin. Eye Res. 18, 463–509
47. van Rijk, A. F., van den Hurk, M. J., Renkema, W., Boelens, W. C., de Jong,
W. W., and Bloemendal, H. (2000) FEBS Lett. 480, 79 – 83
48. Bhattacharyya, J., and Sharma, K. K. (2001) J. Pept. Res. 57, 428 – 434
49. Smulders, R. H., and de Jong, W. W. (1997) FEBS Lett. 409, 101–104
50. Lee, J. S., Satoh, T., Shinoda, H., Samejima, T., Wu, S. H., and Chiou, S. H.
(1997) Biochem. Biophys. Res. Commun. 237, 277–282
51. Santhoshkumar, P., and Sharma, K. K. (2001) Mol. Vis. 7, 172–177
52. Srinivas, V., Datta, S. A., Ramakrishna, T., and Rao, C. M. (2001) Mol. Vis. 7,
114 –119
53. Kumar, L. V., and Rao, C. M. (2000) J. Biol. Chem. 275, 22009 –22013