Critical Review

Chaperone-like Activity and Hydrophobicity of a-Crystallin

G. Bhanuprakash Reddy, P. Anil Kumar and M. Satish Kumar*Biochemistry Division, National Institute of Nutrition, Hyderabad, India

Summary

a-Crystallin, a prominent member of small heat shock protein

(sHsp) family and a major structural protein of the eye lens is a large

polydisperse oligomer of two isoforms, aA- and aB-crystallins.

Numerous studies have demonstrated that a-crystallin functions like

a molecular chaperone in preventing the aggregation of various

proteins under a wide range of stress conditions. The molecular

chaperone function of a-crystallin is thus considered to be vital in

the maintenance of lens transparency and in cataract prevention.

a-Crystallin selectively interacts with non-native proteins thereby

preventing them from aggregation and helps maintain them in a

folding competent state. It has been proposed and generally accepted

that a-crystallin suppresses the aggregation of other proteins

through the interaction between hydrophobic patches on its surface

and exposed hydrophobic sites of partially unfolded substrate

protein. However, a quantifiable relationship between hydrophobi-

city and chaperone-like activity remains a matter to be concerned

about. On an attentive review of studies on a-crystallin chaperone-

like activity, particularly the studies that have direct or indirect

implications to hydrophobicity and chaperone-like activity, we found

several instances wherein the correlation between hydrophobicity and

its chaperone-like activity is paradoxical. We thus attempted to

provide an overview on the role of hydrophobicity in chaperone-like

activity of a-crystallin, the kind of evaluation done for the first time.

IUBMB Life, 58: 632–641, 2006

Keywords sHsp; a-crystallin; crystallin domain; chaperone-likeactivity; hydrophobicity; lens; temperature.

Abbreviations DTT, Dithiothreitol; sHsp, small heat shockproteins; ANS, 1-Anilino-8-naphthalene sulfonicacid; Bis-ANS, 1,10-bi(4-anilino) naphthalene-5,5-disulfonic acid; ITC, Isothermal titrationcalorimetry; G6PD, Glucose-6-phosphate dehydro-genase; GdmCl, Guanidinium hydrochloride.

INTRODUCTION

To become functionally active, nascent protein chains must

fold into unique three-dimensional structures. Although, the

native fold of a protein is encoded in its amino-acid sequence,

protein folding inside cells is not generally a spontaneous

process. Many newly synthesized proteins require complex

cellular machinery comprising of molecular chaperones and an

input of metabolic energy to attain native and thermodynami-

cally stable states. Molecular chaperones are proteins that

assist nascent protein folding and allow the functional state of

proteins to be maintained by preventing irreversible protein

unfolding and aggregation. Numerous studies, over the last

decade have investigated the structural and functional charac-

teristics of molecular chaperones, classifying them into several

families based on size, structure and activity. The small heat

shock proteins (sHsp1) constitute a diverse family of ubiquitous

intracellular proteins. sHsp have subunit masses in the range

of 12 – 43 kDa and exist as large heterogeneous aggregates

of 300 – 1000 kDa that contain 12 – 40 subunits (1). Unlike the

classical chaperones, sHsp prevent the aggregation and

precipitation of a variety of unrelated proteins under stress

conditions such as elevated temperature, reduced and oxidized

conditions at a stoichiometry as low as one subunit of sHsp to

one target protein (1).

a-Crystallin, a major structural protein of the vertebrate eye

lens, is the most intensively studied representative member of

sHsp family. a-Crystallin is known for over a century as one

of the three major crystallins (the other two are b- and

g-crystallins) of the vertebrate eye lens. However, it became a

major focus of studies since 1982, when Drosophila sHsp were

found to share sequence similarities with a-crystallin reflecting

a common evolutionary ancestry (2). Soon after, it was shown

that a-crystallin and other sHsp function like molecular

chaperones in preventing the thermal aggregation of various

proteins including the lens proteins (3, 4). It has been fairly

acknowledged that with aging eye lens proteins undergo

various posttranslational modifications, most of which lead

to aggregation and this process is further accelerated due to

various physiological, environmental and genetic factors that

Received 24 July 2006; accepted 5 September 2006Address correspondence to: G. Bhanuprakash Reddy, National

Institute of Nutrition, Hyderabad – 500 007, India.Fax: þ91 40 27019074. E-mail: geereddy@yahoo.com*Present address: Laboratory of Allergic Diseases, National Instituteof Allergy and Infectious Diseases, National Institutes of Health,USA.

IUBMBLife, 58(11): 632 – 641, November 2006

ISSN 1521-6543 print/ISSN 1521-6551 online � 2006 IUBMB

DOI: 10.1080/15216540601010096

predispose lens to cataract (5). Thus the chaperone-like

function of a-crystallin to suppress the aggregation of proteins

is considered to be critical for the maintenance of lens

transparency. However, the mechanism of chaperone-like

function is not fully understood. It is believed that the first

step in the protective action of a-crystallin is mediated

through the interaction with aggregation-prone or denatured

(non-native) unfolded protein to maintain it in a condition

that favors refolding or prevents from aggregation. One of

the major forces implicated in the mechanistic action of

a-crystallin is entropically driven hydrophobic contacts

between its accessible hydrophobic surfaces and newly exposed

hydrophobic sites of unfolding substrates (6 – 10). Hence, it

is conventionally accepted that hydrophobicity is a major

determinant of chaperone-like activity of a-crystallin.However, upon a careful assessment of the studies on

a-crystallin chaperone-like activity, including studies from

our laboratory, we found numerous instances in which

hydrophobicity alone does not account for the chaperone-like

activity of a-crystallin. In this review we focus on the relation

between hydrophobicity and chaperone-like activity of

a-crystallin.

a-CRYSTALLIN STRUCTURE

In the lens, a-crystallin exists as a heteropolymer with

the molecular size of approximately 800 kDa, having up to

40 subunits from two gene products aA and aB. aA is 173,

while aB is 175 amino acids long and both share 57% sequence

similarity. In most vertebrate lenses, aA and aB subunits exist

in the ratio of 3:1. However the actual ratio varies with species

and also with ageing. Reconstituted as well as recombinant

homopolymers of aA and aB-crystallin are somewhat smaller

(600 – 650 kDa) than the native protein (11, 12), but otherwise

appear to attain similar structural and functional integrity as

that of native a-crystallin. sHsp, including a-crystallin, are

predominantly b-sheet in secondary structure and their

primary sequence can be organized into three distinct struc-

tural regions: an a-crystallin domain of *90 amino acids in

length which is conserved among all sHsps and flanked by

N- and C-terminal domains of variable length and sequence

(Fig. 1). Based on far-UV CD and FT-IR data, secondary

structure of a-crystallin subunits consists of approximately

15% a-helix, 40% b-sheet and the remainder, random coil and

turns (9, 13). The conserved a-crystallin domain spans residues

63 – 144 in aA-crystallin and residues 68 – 148 in aB-crystallin.The amino acid sequence at the a-crystallin domain was shown

to have seven b-strands arranged in two b-sheets; one sheet

consists of three strands and the other of four strands, packed

face to face, forming a sandwich (Fig. 1, 14). Even though,

monomers of many sHsp have the conserved a-crystallindomain, the oligomeric structures display a large variety

between the individual members of the super family. The

poorly conserved N-terminal extension that is highly variable

Figure 1. Alignment of human aA and aB-crystallin amino acid sequences along with human (Hsp27), wheat (Hsp16.9) and

methanococcus jannaschii (Hsp16.5) heat shock proteins. Black bars below the sequences indicate b1 to b10 strands. Consensus

residues are highlighted.

PROPERTIES OF a-CRYSTALLIN 633

in length and hydrophobic in nature influences subunit oligo-

merization and chaperone-like activity whereas the flexible

C-terminal extension stabilizes the global structure and is

thought to enhance protein/substrate complex solubility.

Though, polydispersity of a-crystallin is known to hamper

crystal structure determination, cryo-electron microscopy data

for aB-crystallin indicate it is an irregular spherical structure

with central cavity (15), somewhat similar to that of available

crystal structures of hsp 16.5 and 16.9 (16, 17). Comprehensive

review of structure of a-crystallin and other sHsp has been

covered in (16 – 18).

CHAPERONE-LIKE ACTIVITY OF a-CRYSTALLIN

The chaperone-like activity of a-crystallin and related sHsp

refers to their ability to bind and stabilize non-native protein

substrates and prevent them from subsequent aggregation and

precipitation. Bovine a-crystallin and murine Hsp25 were the

first sHsp reported to have chaperone-like activity (3, 4, 19).

Since then, sHsp from other species have been shown to bind a

variety of non-native proteins in vitro during their chaperon-

ing-like function. The chaperone-like activity of sHsp are

usually assayed by simple in vitro aggregation experiments in

which substrate proteins are subjected to aggregation by heat

or chemical treatment in the presence of the chaperone

(19 – 24). The extent of aggregation of substrate protein is

usually measured in terms of optical density. DTT-induced

aggregation of insulin (due to precipitation of insulin B-chain

upon reduction) and heat-induced aggregation of citrate

synthase (42 – 508C) or b-crystallin (*608C) are widely

employed assays, though numerous other assays have been

used, for assessing the chaperone like function of sHSP.

However, it should be noted that the conclusions from

these experiments might not always be applicable to in vivo

situations (21). Typically in these studies it is assumed that the

chaperone itself is unaffected by the treatment used for

substrate aggregation. In contrast to other classes of molecular

chaperones, each oligomeric sHsp complex binds several non-

native polypeptide chains. Though ratios of up to one non-

native substrate protein per subunit have been reported, this

may vary. The substrate specificity of a-crystallin is rather

broad; chaperone-like activity has been demonstrated in vitro

with various lenticular and non-lenticular proteins against

physical and physiological stress such as heat, UV irradiation,

chemical stress, oxidative stress and glycation (3, 4, 6, 9,

20 – 25). Although, a-crystallin has been shown to facilitate

refolding of several proteins and enzymes, for example citrate

synthase, quinone oxidoreductase, lysozyme, ribonuclease,

xylose reductase and glucose-6-phosphate dehydrogenase

(G6PD) (26 – 28), the degree to which enzyme activity is

regained upon refolding remains inconclusive.

Chaperone-like activity of endogenous a-crystallin in main-

taining lens transparency was clearly evident in experiments

where the total soluble lens homogenate subjected to heat

showed little aggregation (29 – 31). On the other hand, lens

homogenate that is devoid of a-crystallin and subjected to heat

showed increased aggregation (31). The physiologic signifi-

cance of the chaperone function of a-crystallin is further

substantiated by the findings that mutations in a-crystallinthat are known to cause cataract have a dramatic effect on its

chaperone-like activity as well (reviewed in 1) (Table 1).

A missense mutation (R120G) in aB-crystallin is shown to

cause a familial form of desmin-related myopathy (DRM)

characterized by intracellular desmin aggregation, cardiomyo-

pathy and cataract (32). A similar mutation in aA-crystallin

(R116C) is shown to cause cataract (33). Recombinant

proteins with these mutations displayed altered physicochem-

ical properties with a considerably diminished ability to

suppress protein aggregation (discussed later). Animal knock-

outs, either aA or aB or both, provided further insights into

the physiologic relevance of a-crystallin chaperone-like func-

tion (reviewed in 20). Interestingly, while aA-crystallin knock-

out mice developed cataract, aB knockout mice showed no

signs of cataract but degeneration of skeletal muscles. Though,

a-crystallin is a heteropolymer of aA and aB, it is interesting tonote that phenotypes are different with aA and aB knockouts.

Furthermore, primary cultures of lens epithelial cells derived

from aB knockouts showed genome instability and hyper

proliferation suggesting that these proteins may also have an

important role to play in the maintenance and regulation of

genomic stability and cell division (34). Thus, these studies

implicate a role for aA- and aB-crystallins not only in the

maintenance of lens transparency but also in various other

cellular functions (35).

HYDROPHOBICITY AND CHAPERONE-LIKE ACTIVITY

While the chaperone-like activity of a-crystallin has been

proven to be critical under stress conditions, the molecular

mechanism of this activity remains largely unknown.

Understanding the molecular forces involved in the interaction

of a-crystallin with the substrate proteins is very important to

get insights into the mechanism by which a-crystallin protects

proteins against various physical and biochemical assaults.

Several sites on a-crystallin have been postulated to be

involved in the chaperone-like function and complex forma-

tion, including the N-terminal and C-terminal regions. The

C-terminal region of aA-crystallin (36) and the mostly

hydrophobic N-terminal phenylalanine-rich region in

aB-crystallin have been suggested to be necessary for

chaperone-like activity (37). Sharma et al. reported that

residues 57 – 69 and 93 – 107 in aB-crystallin are involved in

chaperoning alcohol dehydrogenase (38). Later they have also

reported that residues 50 – 54 and 79 – 99 in aA-crystallin and

residues 75 – 103 in aB-crystallin are bis-ANS-binding and

concluded that the hydrophobic sites participate in chaperone-

like activity of the protein (38 – 40). Based on protein pin

arrays, Ghosh et al. identified seven putative substrate

634 REDDY ET AL.

interactive sites in aB-crystallin that include two sequences

from the N-terminal domain, four sequences from the

a-crystallin domain and one from the C-terminal domain

(41). Five of these interactive sequences are identified as

sequences associated with subunit-subunit interactions in

human aB-crystallin. Based on these studies it appears that

virtually every region of a-crystallin is vital for its chaperone-

like function. Further, involvement of variable sequence

outside the a-crystallin domain in substrate binding suggests

that substrate-binding profiles may vary among different

sHsps and it may also depend on the arrangement of subunits

in three-dimensional conformation.

Irrespective of the involvement of specific sequence elements

that mediate either substrate binding or oligomerization,

several studies on a-crystallin have attributed chaperone-like

function to its hydrophobic surfaces interacting with the

exposed hydrophobic sites of denaturing substrate proteins.

Denaturing substrates expose buried hydrophobic surfaces

as a result of their unfolding and this was expected to

facilitate either self-aggregation or interaction with sHsp.

Table 1

Effect of site-directed mutations on �-crystallin chaperone activity that have a bearing (directly or indirectly) onhydrophobicity. Chaperone activity and hydrophobicity is compared with wild type protein

Protein Mutation

Chaperone

activity Hydrophobicity Remark Ref

�B (Mouse) D2G

F24R

Decreased

Abolished

Polar residue is substituted by non-polar

residue at N-terminal end of protein

(37)

F27R Abolished

F27A

K174L/K175L

Abolished

No significant

Mutations in phenyl alanine region abolish

chaperone activity

K174G/K175G activity Insertion of hydrophobic residues in place of

charged residues in C-terminal region resulted

in loss of chaperone activity

�B (Human) F27R No change Substitution of hydrophobic phenylalanine with

arginine has no effect on chaperone activity

(70)

�A (Rat) F71G Decreased Increased Substitution of hydrophobic phenyl alanine with

glycine resulted in loss of chaperone activity,

while hydrophobicity was increased

(56)

�B (Mouse) F118A

K103L/H104I

E110H/H111E

No change K103L/H104I double mutant display identical

chaperone activity as wild type protein despite

incorporating hydrophobic residues

(57)

�B M68I

M68V

M68T

Increased

Increased

Decreased

While insertion of hydrophobic residues

increased chaperone activity, hydrophilic

residues decreased the activity in the putative

target protein binding site

(72)

�A R116C

R116G

R116D

Decreased

Decreased

Significant

loss

Substitution of conserved arginine with aspartic

acid resulted in the significant loss of

chaperone activity. Positive charge at this

position appears to be critical

(67)

�B (Human) R120G Decreased Positive charge at this position appears to be

critical for chaperone activity

(66)

�A (Bovine) D69S Decreased No change Mutation D69S resulted in loss of chaperone

activity without a change in hydrophobicity

(71)

�B S66G No loss Increased S66G mutant displayed increased

hydrophobicity where as chaperone

activity is identical to wild type

(73)

PROPERTIES OF a-CRYSTALLIN 635

These sHsp-bound substrates are further refolded to their

native state by ATP dependent chaperones, such as Hsp70/

Dna K. Thus, sHsp appear to function like a reservoir of

partially unfolded proteins in refolding-competent state

(Fig. 2). In the case of classical chaperones, such as E. coli

GroEL, the chaperone activity has been attributed to surface

hydrophobicity of both the chaperone and its substrates (42).

Similarly, chaperone-like activity of 70S ribosome was

attributed to its surface hydrophobicity (43). Atp11p, a soluble

mitochondrial chaperone, is also known to prevent F1-ATPase

aggregation in matrix through hydrophobicity mediated

chaperone-substrate interactions (44). Thus it is conceivable

that hydrophobicity is a major factor in the chaperoning

function of many chaperones including a-crystallin. This is

substantiated by the experimental data that indicate that

a-crystallin preferentially recognizes non-native structures,

which are likely to have increased surface hydrophobicity on

the denaturation pathway (45). However, the so-called hydro-

phobic ‘non-native structures’ also seem to be structurally

diverse entities (46). The studies with three non-identical non-

native states of lactalbumin suggest that a-crystallin interacts

only with the aggregation prone molten globule state of

reduced apo-a-lactalbumin but not with the other non-

aggregating molten globule states of the protein (46). It has

been shown that a-crystallin specifically recognizes very early

intermediates on the denaturation pathway of proteins, which

are characterized by native-like secondary structure but

compromised tertiary interactions (47, 48). The structural state

of zeta-crystallin that binds to a-crystallin was shown to be a

partially unfolded inactive monomer that exhibits highly

exposed hydrophobic surfaces and has significant secondary

structural elements with little or no tertiary structure (49). This

intermediate does not refold into the active state without

assistance and a-crystallin provides the required assistance and

improves the reactivation yield several-fold. The strongest

supporting evidence for the involvement of hydrophobicity in

chaperone-like function came from temperature-dependent

experiments. Upon heating, a-crystallin undergoes structural

changes resulting in increased exposure of additional hydro-

phobic sites associated with increased chaperone-like activity

(6, 7, 9, 10, 47, 50). The chaperone-like activity of a-crystallin is

less pronounced at 308C as compared to its activity at 558C(51). While there is a minor alteration in a-crystallin tertiary

structure at 308C, above 508C, it undergoes a structural transi-tion to a molten globule-like state that has increased hydro-

phobic surfaces. More importantly when a-crystallin is heated

to high temperatures and then cooled (preheated), not only

there is a dramatic increase in surface hydrophobicity, the

a-crystallin exhibited a significant increase in chaperone-like

activity as compared to unheated a-crystallin (7, 9, 10). Once

exposed to high temperatures, the protein upon cooling does

not return to its original conformational state but adopts a

conformation characterized by significantly increased surface

hydrophobicity (7). Structural perturbations due to factors

Figure 2. A schematic mechanism of chaperone-like function of a-crystallin. a-Crystallin selectively interacts with non-native

intermediates on their unfolding pathway and stabilizes them in a refolding competent state. Hydrophobic forces may mediate

the interaction between the chaperone and partially unfolded substrate.

636 REDDY ET AL.

other than heat also enhanced the chaperone-like activity,

which were associated with increased hydrophobicity. Arginine

hydrochloride brings about subtle changes in the tertiary

structure and significant changes in the quaternary structure of

a-crystallin that resulted in enhanced chaperone-like activity,

which is mediated through increased exposure of hydrophobic

surfaces (52). In the guanidine hydrochloride (GdnHCl)

induced unfolding pathway; a molten globule-like intermediate

of a-crystallin was shown to have maximum chaperone-like

activity concurrent with maximum hydrophobicity (53). These

studies are considered as ‘‘gold standards of evidence’’ of the

intimate relationship between hydrophobicity and chaperone-

like activity of a-crystallin.However, despite a wealth of evidence for dependence of

the chaperone function of a-crystallin on its surface hydro-

phobicity, a deterministic role of hydrophobicity in quantita-

tive terms remains unresolved. There are numerous instances

where hydrophobicity has not been directly correlated to the

chaperone-like activity of either a-crystallin heteropolymer or

aA or aB-homopolymers. Though it is strongly believed that

the a-crystallin domain contributes to hydrophobicity and is

associated with chaperone-like activity, sequence alignment

does not indicate the presence of either conserved hydro-

phobic sequences or conserved hydrophobic residues (Fig. 1).

Perhaps the ‘so called’ hydrophobic surfaces might appear

after attaining the global conformation. This may be the

reason for exhibiting increased hydrophobicity upon reorga-

nization after structural perturbations. Further, it was shown

that contacts between a conserved motif (with the sequence I-

X-I/V) in the C-terminal region and a hydrophobic patch in

the a-crystallin domain of neighboring subunits are critical for

oligomer formation (54). This raises the question as to how the

hydrophobic domain of a-crystallin mediates two different

events, oligomerization and substrate binding. It is possible

that subunits may come apart to bind the substrate but it

is not straightforward to determine the hydrophobicity of

a-crystallin under those circumstances. Although, the heat-

shock domain of a-crystallin is implicated in chaperone-

like function, it has been reported that this domain has no

chaperone-like activity independently (55). Further, the

excised heat-shock domain is found to be predominantly

trimeric and displays significant surface hydrophobicity and a

tendency to undergo self-aggregation. It suggests that surface

hydrophobicity may alone be insufficient for this domain to

function as a chaperone (55).

Replacement of Phe71 with Gly in aA-crystallin results

in the loss of chaperone-like activity despite an increase in

surface hydrophobicity with no significant alteration in

structure (56). This is an interesting example where hydro-

phobicity does not correspond with chaperone-like activity.

Residues H101 to R120 are highly conserved between aA- and

aB-crystallin. However, single mutation (F118A) and double

mutations (K103L/H104I) did not affect the chaperone-like

activity of aB-crystallin, in spite of the replacement of

hydrophobic residues. (57). Using insulin aggregation assay,

all three mutants had identical chaperone-like activity to the

wild-type recombinant aB-crystallin. Likewise, insertion of

hydrophobic residues into the C-terminal extension of aA-

crystallin results in a partial loss of the flexibility in this region

and a concomitant decrease in chaperone-like activity and

stability of the protein (58). Studies showed that high mole-

cular weight (HMW) complexes of a-crystallin with its

substrate formed during heat induced aggregation exhibited

a greater hydrophobicity than complexes formed during DTT-

induced aggregation (59), regardless of its similar ability to

suppress the aggregation under both the conditions. Based on

these observations, in contrast to the existing impression, it

appears that hydrophobicity may not play a central role in

chaperone-like activity of a-crystallin. Some studies have

attempted to highlight the importance of hydrophobicity by

incorporation of ANS or bis-ANS, which is expected to block

the hydrophobic sites. But these studies indicate only a partial

loss of chaperone-like activity upon blocking the surface

hydrophobicity (39, 60). This is surprising, because one would

expect a loss in chaperone-like activity with prior ANS

binding. Further, if chaperone-like activity is predominantly

due to hydrophobicity, the loss in chaperone-like activity with

prior ANS binding could be much greater under conditions

that enhance the chaperone activity. For instance, the increase

in chaperone-like activity was much greater with preheated aAas compared to aB, which is perceived to be due to higher

hydrophobicity, but the percentage loss of chaperone-like

activity due to ANS binding was similar, with these preheated

aA- and aB-crystallins (10).a-Crystallin modified with methlyglyoxal (MGO), a highly

reactive dicarbonyl cross linking agent, exhibited increased

chaperone-like activity in conventional aggregation assays

despite an apparent decrease in hydrophobicity due to MGO

modification (21). This is another example, where chaperone-

like activity and hydrophobicity run exactly in opposite

directions. Yet, MGO-modified a-crystallin’s ability to prevent

inactivation of enzymes was compromised as compared to

native a-crystallin (21). Clearly further investigation is needed

to determine whether the ability of a-crystallin in protecting

the enzymes is independent of hydrophobicity. Interestingly

aB-crystallin specifically assisted the refolding and reactivation

of completely unfolded G6PD by GdmCl where as it is unable

to assist the reactivation of molten globule-like state that is

highly hydrophobic intermediate in GdmCl-induced unfolding

of G6PD (27). In the case of xylose reductase, a-crystallin was

shown to interact specifically with its molten globule state in

reconstituting the enzyme activity (28). While the molten

globule state of a protein is richly hydrophobic, the interaction

of a-crystallin with molten globule species of different sub-

strates appears to be different (27, 28).

Although, temperature dependent experiments suggest the

involvement of hydrophobicity in the chaperone-like activity

of a-crystallin, the relationship between hydrophobicity and

PROPERTIES OF a-CRYSTALLIN 637

increased chaperone-like activity at elevated temperatures

differs in the case of different species of a-crystallins (9 – 12, 47,50). At physiological temperatures (25 – 378C) recombinant aBdisplayed higher chaperone-like activity than recombinant aA-

crystallin and aB appears to be more hydrophobic than aA at

these temperatures (9). At high temperatures where the hydro-

phobicity of recombinant aA increases and aB decreases (9),

recombinant aA-crystallin shows higher chaperone-like activ-

ity than recombinant aB. In contrast, while reconstituted

aA-crystallin (from calf a-crystallin) exhibits slightly increased

hydrophobic profile than the reconstituted aB at room tem-

perature, the chaperone-like activity of aA-crystallin is lower

than that of aB-crystallin (12). Bis-ANS binding to both

reconstituted aA- and aB-crystallin decrease with increase in

temperature despite enhanced chaperone potential (12). When

hydrophobicity of aA and aB-crystallin was analyzed by Kyte-

Doolittle plots that characterize hydrophobic nature based on

protein sequence, it predicts that aA is more hydrophobic than

aB-crystallin (Fig. 3). Therefore chaperone-like activity of

a-crystallin does not appear to be quantitatively related to

their hydrophobicity and studies that indicate a correlation

between hydrophobicity and chaperone-like activity of

a-crystallin, either positive or paradoxical, might be a simple

coincidence.

Very few studies have determined hydrophobic sites of a-crystallin quantitatively. These studies, based on fluorescence

emission, used the two common fluorescence probes, ANS and

bis-ANS. The results of these studies are however not in

agreement with each other. For instance, one study reported

that there are about 40 ANS binding sites per native

a-crystallin (61). Another study has disputed the above study

and reported that there is one ANS-binding site per 24

subunits of a-crystallin (62). Others have reported one ANS or

bis-ANS binding site per subunit of a-crystallin (8, 9).

Moreover, the polydisperse nature of a-crystallin may com-

plicate the quantification of hydrophobicity by spectroscopic

methods. More sensitive and accurate methods are needed to

resolve such ambiguities. Recently we quantified the hydro-

phobicity, using high sensitivity isothermal titration calorime-

try (ITC), directly as the number of ANS binding sites on

recombinant aA and aB-crystallins (10). Both aA- and

aB-crystallin showed two modes of binding for ANS: low-

affinity and high-affinity. Thermodynamic parameters for the

binding of ANS to aA- and aB-crystallin indicate that high

affinity binding is driven by both enthalpy and entropy

changes, whereas the entropy change is dominant for low

affinity binding. aA-Crystallin, at 308C showed one high

affinity binding site per subunit for ANS, and a large number

(eighteen) of low affinity binding sites. There were more high

and low affinity sites in aB (six and twenty seven respectively)

as compared to aA at 308C. Thus, one can argue that aB is

more hydrophobic than aA at room temperature (308C). As

expected, at room temperature aB-crystallin showed higher

chaperone-like activity than aA-crystallin consistent with the

greater number of ANS binding sites (both high and low

affinity) (10). Though many studies attributed increased

chaperone-like activity at higher temperature to increased

hydrophobicity, by ITC method we found similar ANS

binding sites for both aA and aB (10). In addition, we have

also investigated chaperone-like activity and hydrophobicity

at low temperatures to get more insights of their association.

Interestingly, while ANS binding sites were found to be similar

in number for both aA and aB at 158C (high affinity- 3.5 and

3.1; low affinity- 12.2 and 11.9 respectively for aA and aB), aAshowed relatively greater chaperone-like activity than aBcrystallin at this temperature (10). Intriguingly, aA showed

more high affinity ANS binding sites at 158C compared to

308C (3.5 vs 0.98), but its ability to suppress insulin

aggregation was much lower at 158C than at 308C. Together,these studies indicate that relative chaperone-like activities of

aA and aB are not correlated with the number of hydro-

phobic sites.

Apart from hydrophobicity several other factors could

influence the chaperone-like activity of a-crystallin. These

include oligomeric size/state, subunit exchange, quaternary

structure and stability of a-crystallin as well as ionic inter-

actions between the chaperone and substrate. The increase in

chaperone-like activity of a-crystallin at higher temperature

(above 508C) could also be attributed to the formation of high

molecular weight aggregates and altered secondary structure.

It has been suggested that chaperone-like activity depends in

part on the packing parameters of the aggregate and on

conformation of the subunit within that aggregate (63). In this

Figure 3. Kyte-Doolittle plot of hydrophobicity of human aA-

(Panel A) and aB-crystallin (Panel B).

638 REDDY ET AL.

regard, methylglyoxal modified a-crystallin that has larger

aggregate size showed increased chaperone-like activity

despite a decrease in surface hydrophobicity (21). Remark-

ably, however, sHsps were also found to exhibit increased

chaperone-like activities while maintaining their oligomeric

size unchanged. sHsp complexes are dynamic in structure and

exchange subunits constantly and this appears to be one of the

properties of sHsps that is important for their activity (64).

The dynamic behavior of sHsp could allow the substrate-

binding sites, which are normally buried in the oligomeric

complex, to become exposed on dissociation. This led to the

hypothesis that dissociation of the oligomer is required for

recognition of the substrate. It has been demonstrated that

subunit exchange in a-crystallin is a key factor in chaperone-

like function (65). It should be noted that both oligomeriza-

tion and subunit exchange are interconnected and mediated by

hydrophobic interactions. The conserved residues such as

R112 and R116 in the buried region of the protein form salt

bridges between the subunits (66). Studies with various

recombinant constructs of R116 of aA-crystallin (R116K,

R116G, R116C and R116D) and R120 of aB-crystallin(R120G) have shown that a positive charge must be preserved

at this position for structural and functional integrity of

a-crystallin (66, 67). In addition, presence of charged residues

at the C-terminal end of a-crystallin has been shown to be

critical for solvent interaction. Hence, disruption of net charge

of the protein may affect structural stability and chaperone-

like activity.

In summary, there are several factors that seem to influence

chaperone-like activity, hydrophobicity may be one of the

factors but may not be the predominant one. Further, chaper-

oning function and mechanism may vary depending on the

substrate and other prevailing conditions. Thus, proposing a

universal mechanism for the chaperone-like activity of

a-crystallin or sHsp may not be feasible.

FUTURE DIRECTIONS

Apart from its presence in the lens, a-crystallin is also

found in many non-lenticular tissues, albeit at very low levels

(as reviewed in ref 68). Furthermore, elevated expression of

a-crystallin, particularly aB-crystallin, has been observed in

many pathological conditions (1, 68, 69). Although, the

importance of a-crystallin in non-lenticular tissues is acknowl-

edged merely as stress protein, its function in non-lenticular

tissues still remains unanswered. Investigating the mechanism

of a-crystallin mediated cellular functions, particularly in non-

lenticular tissues, such as cell differentiation and cytoskeletal

organization could throw a light on the significance of hydro-

phobicity regarding chaperone-like activity. It may be noted

that while aA-crystallin is present in a limited number of

tissues at very low levels, aB-crystallin is prominent in quite a

few non-lenticular tissues. However, a-crystallin has not been

purified from any tissues other than lens. Moreover, whether it

exists as an aggregate (oligomer) or some other quaternary

state in other tissues remains to be answered. Thus, studies

should focus on characterizing structural and functional

aspects with respect to hydrophobicty of a-crystallin purified

from non-lenticular tissues. At the same time, understanding

the significance of the existence of two subunits of a-crystallinas a heteropolymer with 3:1 ratio in the lens may also provide

further insights into hydrophobicity and chaperone-like

function. A serious deficiency in the studies that relate the

chaperone-like activity and hydrophobicity of a-crystallin is

that in these studies the researchers have used different pre-

parations of a-crystallin (e.g., native a-crystallin hetero-

polymer, recombinant or reconstituted homopolymers of aAor aB) and diverse chaperone assays with various substrates.

It is known that physicochemical properties of a-crystallin can

vary depending on the sources (e.g., young or old lens,

species), the purification methods employed, the presence of

posttranslational modifications and the chaperone assay

conditions. Hence, assessing source and purification of

a-crystallin and the probes used for measuring hydro-

phobicity is of great concern in relating the hydrophobicity

to chaperone-like function. Thus, studies are warranted to

resolve the hydrophobicity paradox in order to provide greater

insights into chaperone-like activity of a-crystallin.

ACKNOWLEDGEMENTS

Grants from Department of Science and Technology, Govern-

ment of India and Indian Council of Medical Research,

Government of India to GBR are acknowledged. The authors

thank Prof. A. Surolia, Indian Institute of Science, Bangalore

for the use of their biophysical facilities and critical comments

and Mr. P. Yadagiri Reddy for his help at various aspects of

the work reported in this review. PAK and MSK acknowledge

Council of Scientific and Industrial Research for providing

research fellowship.

REFERENCES1. Sun, Y., and MacRae, T. H. (2005) The small heat shock proteins and

their role in human disease. FEBS J. 272, 2613 – 2627.

2. Ingolia, T. D., and Craig, E. A. (1982) Four small Drosophila heat

shock proteins are related to each other and to mammalian

a-crystallin. Proc. Natl. Acad. Sci. USA 79, 2360 – 2364.

3. Horwitz, J. (1992) Alpha-crystallin can function as a molecular

chaperone. Proc. Natl. Acad. Sci. USA 89, 10449 – 10453.

4. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) Small heat

shock proteins are molecular chaperones. J. Biol. Chem. 268,

1517 – 1520.

5. Harding, J. J. (1991) In Cataract; Biochemistry, Epidemiology and

Pharmacology, Chapman & Hall, London.

6. Raman, B., and Rao, C. M. (1994) Chaperone-like activity

and quaternary structure of alpha-crystallin. J. Biol. Chem. 269,

27264 – 27268.

7. Das, K. P., and Surewicz, W. K. (1995) Temperature-induced

exposure of hydrophobic surfaces and its effect on the chaperone

activity of alpha-crystallin. FEBS Lett. 369, 321 – 325.

PROPERTIES OF a-CRYSTALLIN 639

8. Sharma, K. K., Kaur, H., Kumar, G. S., and Kester, K. (1998)

Interaction of 1,10-bi(4-anilino) naphthalene-5, 50-disulfonic acid with

alpha-crystallin. J. Biol. Chem. 273, 8965 – 8970.

9. Reddy, G. B., Das, K. P., Petrash, J. M., and Surewicz, W. K. (2000)

Temperature-dependent chaperone activity and structural properties

of human alphaA- and alphaB-crystallins. J. Biol. Chem. 275,

4565 – 4570.

10. Kumar, M. S., Kapoor, M., Sinha, S., and Reddy, G. B. (2005)

Insights into hydrophobicity and the chaperone-like function of

alphaA- and alphaB-crystallins: an isothermal titration calorimetric

study. J. Biol. Chem. 280, 21726 – 21730.

11. Sun, T. X., Das, B. K., and Liang, J. J. (1997) Conformational and

functional differences between recombinant human lens alphaA- and

alphaB-crystallin. J. Biol. Chem. 272, 6220 – 6225.

12. Bhattacharyya, J., Srinivas, V., and Sharma, K. K. (2002) Evaluation

of hydrophobicity versus chaperone like activity of bovine aA and

aB-crystallin. J. Protein Chem. 21, 65 – 71.

13. Farnsworth, P. N., Groth-Vasselli, B., Greenfield, N. J., and Singh, K.

(1997) Effects of temperature and concentration on bovine lens

alpha-crystallin secondary structure: a circular dichroism spec-

troscopic study. Int. J. Biol. Macromol. 20, 283 – 291.

14. Koteiche, H. A., and Mchaourab, H. S. (1999) Folding pattern of

the alpha-crystallin domain in alphaA-crystallin determined by site-

directed spin labeling. J. Mol. Biol. 294, 561 – 577.

15. Haley, D. A., Horwitz, J., and Stewart, P. L. (1998) The small heat-

shock protein, alphaB-crystallin, has a variable quaternary structure.

J. Mol. Biol. 277, 27 – 35.

16. Van Montfort, R., Slingsby, C., and Vierling, E. (2001) Structure and

function of the small heat shock protein/alpha-crystallin family of

molecular chaperones. Adv. Protein Chem. 59, 105 – 156.

17. Augusteyn, R. (2004) a-crystallin: a review of its structure and

function. Clin. Exp. Optom. 87, 356 – 366.

18. Carver, J. A., and Linder, R. A. (1998) NMR spectroscopy of alpha-

crystallin. Insights into the structure, interactions and chaperone

action of small heat shock proteins. Int. J. Biol. Macromol. 22,

197 – 209.

19. Merck, K. B., Groenen, P. J. T. A., Voorter, C. E., de Haard-

Hoekman, W. A., Horwitz, J., Bloemendal, H., and dejong, W. W.

(1993) Structural and functional similarities of bovine a-crystallin and

mouse small heat shock proteins. J. Biol. Chem. 268, 1046 – 1052.

20. Horwitz, J. (2003) Alpha-crystallin. Exp. Eye Res. 76, 145 – 153.

21. Kumar,M. S., Reddy, P.Y.,Kumar, P.A., Surolia. I., andReddy,G.B.

(2004) Effect of dicarbonyl-induced browning on alpha-crystallin

chaperone-like activity: physiological significance and caveats of

in vitro aggregation assays. Biochem. J. 379, 273 – 282.

22. Reddy, G. B., Narayanan, S., Reddy, P. Y., and Surolia, I. (2002)

Suppression of DTT-induced aggregation of abrin by alphaA- and

alphaB-crystallins: a model aggregation assay for alpha-crystallin

chaperone activity in vitro. FEBS Lett. 522, 59 – 64.

23. Hook, D. W., and Harding, J. J. (1998) Protection of enzymes by

alpha-crystallin acting as a molecular chaperone. Int. J. Biol.

Macromol. 22, 295 – 306.

24. Reddy, G. B., Reddy, P. Y., and Suryanarayana, P. (2001) aA- and

aB-crystallins protect glucose-6-phosphate dehydrogenase against

UVB irradiation-induced inactivation. Biochem. Biophys. Res.

Commun. 282, 712 – 716.

25. Hess, J. F., and FitzGerald, P. G. (1998) Protection of a restriction

enzyme from heat inactivation by [alpha]-crystallin. Mol. Vis. 4, 29.

26. Raman, B., Ramakrishna, T., and Rao, C. M. (1997) Effect of the

chaperone-like alpha-crystallin on the refolding of lysozyme and

ribonucleaseA. FEBS Lett. 416, 369 – 372.

27. Kumar, M. S., Reddy, P. Y., Sreedhar. B., and Reddy, G. B. (2005)

Alphab-crystallin-assisted reactivation of glucose-6-phosphate dehy-

drogenase upon refolding. Biochem. J. 391, 335 – 341.

28. Rawat, U., and Rao, M. (1998) Interactions of chaperone

alpha-crystallin with the molten globule state of xylose reductase.

Implications for reconstitution of the active enzyme. J. Biol. Chem.

273, 9415 – 9423.

29. Rao, P. V., Huang, Q. L., Horwitz, J., and Zigler, J. S. Jr (1995)

Evidence that alpha-crystallin prevents non-specific protein aggrega-

tion in the intact eye lens. Biochim. Biophys. Acta 1245, 439 – 447.

30. Reddy, G. B., Reddy, P. Y., Vijayalakshmi, A., Kumar, M. S.,

Suryanarayana, P., and Sesikeran, B. (2002) Effect of long-term

dietary manipulation on the aggregation of rat lens crystallins: role of

alpha-crystallin chaperone function. Mol. Vis. 8, 298 – 305.

31. Horwitz, J. (1993) Proctor Lecture. The function of alpha-crystallin.

Invest. Ophthalmol. Vis. Sci. 34, 10 – 22.

32. Vicart, P., Caron, A., Guicheney, P., Li, Z., Prevost, A. F.,

Chatean, D., Chapon, F., Tome, F., Dupret, J. M., and Saulin, D.

(1998) A missense mutation in the alphaB-crystallin gene causes a

desmin related myopathy. Nature Gen. 20, 92 – 95.

33. Litt,M.,Kramer, P., LaMorticella,D.M.,Murphy,W., Lovrien,E.M.,

and Weleber, R. G. (1998) Autosomal dominant congenital cataract

associated with a missense mutation in the human alpha gene CRYAA.

Hum. Mol. Genet. 7, 471 – 474.

34. Andley, U. P., Song, Z., Wawrousek, E. F., Brady, J. P, Bassnett, S.,

and Fleming, T. P. (2001) Lens epithelial cells derived from

alphaB-crystallin knockout mice demonstrate hyperproliferation and

genomic instability. FASEB J. 15, 221 – 229.

35. Boyle, D. L., Takemoto, L., Brady, J. P., and Wawrousek, E. F.

(2003) Morphological characterization of the Alpha A- and Alpha

B-crystallin double knockout mouse lens. BMC Ophthalmol. 3, 3.

36. Takemoto, L., Emmons, T., and Horwitz, J. (1993). The C-terminal

region of alpha-crystallin: involvement in protection against heat-

induced denaturation. Biochem. J. 294, 435 – 438.

37. Plater, M. L., Goode, D., and Crabbe. M. J. (1996) Effects of site-

directed mutations on the chaperone-like activity of alphaB-crystallin.

J. Biol. Chem. 271, 28558 – 28566.

38. Sharma, K. K., Kaur, H., and Kester, K. (1997) Functional elements

in molecular chaperone alpha-crystallin: identification of binding sites

in alpha B-crystallin. Biochem. Biophys. Res. Commun. 239, 217 – 222.

39. Sharma, K. K., Kumar, G. S., Murphy, A. S., and Kester, K. (1998)

Identification of 1,10-bi(4-anilino) naphthalene-5, 50-disulfonic acid

binding sequences in alpha-crystallin. J. Biol. Chem. 273,

15474 – 15478.

40. Sharma, K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000)

Synthesis and characterization of a peptide identified as a functional

element in alphaA-crystallin. J. Biol. Chem. 275, 3767 – 3771.

41. Ghosh, J. G., Estrada, M. R., and Clark, J. L. (2005) Interactive

domains for chaperone activity in the small heat shock protein, human

alphaB crystallin. Biochemistry 44, 14854 – 14869.

42. Melkani, G. C., Zardeneta, G., and Mendoza, J. A. (2004) Oxidized

GroEL can function as a chaperonin. Front Biosci. 9, 724 – 731.

43. Singh, R., and Rao, Ch. M. (2002) Chaperone-like activity and

surface hydrophobicity of 70S ribosome. FEBS Lett. 527, 234 – 238.

44. Sheluho, D., and Ackerman, S. H. (2001) An accessible hydrophobic

surface is a key element of the molecular chaperone action of Atp11p.

J. Biol. Chem. 276, 39945 – 39949.

45. Das, K. P., Petrash, J. M., and Surewicz, W. K. (1996) Conforma-

tional properties of substrate proteins bound to a molecular

chaperone a-crystallin. J. Biol. Chem. 271, 10449 – 10452.

46. Rajaraman, K., Raman, B., Ramakrishna, T., and Rao, C. M. (1998)

The chaperone-like alpha-crystallin forms a complex only with the

aggregation-prone molten globule state of alpha-lactalbumin.

Biochem. Biophys. Res. Commun. 249, 917 – 921.

47. Datta, S. A., and Rao, C. M. (1999) Differential temperature-

dependent chaperone-like activity of aA- and aB-crystallin homo-

aggregates. J. Biol. Chem. 274, 34773 – 34778.

640 REDDY ET AL.

48. Das, K. P., Choo-Smith, L. P., Petrash, J. M., and Surewicz, W. K.

(1999) Insight into the secondary structure of non-native proteins

bound to a molecular chaperone alpha-crystallin. An isotope-edited

infrared spectroscopic study. J. Biol. Chem. 274, 33209 – 33212.

49. Goenka, S., Raman, B., Ramakrishna, T., and Rao, C. M. (2001)

Unfolding and refolding of a quinone oxidoreductase: alpha-

crystallin, a molecular chaperone, assists its reactivation. Biochem.

J. 359, 547 – 556.

50. van Boekel, M. A., de Lange, F., de Grip, W. J., and de Jong, W. W.

(1999) Eye lens alphaA- and alphaB-crystallin: complex stability

versus chaperone-like activity. Biochim. Biophys. Acta 1434, 114 – 123.

51. Raman, B., and Rao, C. M. (1997) Chaperone-like activity and

temperature-induced structural changes of alpha-crystallin. J. Biol.

Chem. 272, 23559 – 23564.

52. Srinivas, V., Raman, B., Rao, K. S., Ramakrishna, T., and

Rao, Ch. M. (2003) Structural perturbation and enhancement of the

chaperone-like activity of alpha-crystallin by arginine hydrochloride.

Protein Sci. 12, 1262 – 1270.

53. Das, B. K., and Liang, J. J. (1997) Detection and characterization of

alpha-crystallin intermediate with maximal chaperone-like activity.

Biochem. Biophys. Res. Commun. 236, 370 – 374.

54. Pasta, Y., Raman, B., Ramakrishna, T., and Rao, C. M. (2004) The

IXI/V motif in the C-terminal extension of alpha-crystallins: alter-

native interactions and oligomeric assemblies. Mol. Vis. 10, 655 – 662.

55. Kundu, B., Shukla, A., Chaba, R., and Guptasarma, P. (2004) The

excised heat-shock domain of alphaB crystallin is a folded,

proteolytically susceptible trimer with significant surface hydropho-

bicity and a tendency to self-aggregate upon heating. Protein Expr.

Purif. 36, 263 – 271.

56. Kumar, P. S., and Sharma, K. K. (2001) Phe71 is essential for

chaperone-like function in alpha A-crystallin. J. Biol. Chem. 276,

47094 – 47099.

57. Hepburne-Scott, H. W., and Crabbe, M. J. C. (1999) Maintenance of

chaperone like activity despite mutations in a conserved region of

murine lens aB crystallin. Mol. Vis. 5, 15.

58. Smulders, R.H. P. H., Carver, J. A., Lindner, R. A., van Boekel,M. A.,

Bloemendal, H., and de Jong, W. W. (1996) Immobilization of the

C-terminal extension of bovine alphaA-crystallin reduces chaperone-

like activity. J. Biol. Chem. 271, 29060 – 29066.

59. Lindner,R.A.,Kapur,A.,Mariani,M., Titmuss, S. J., andCarver, J.A.

(1998) Structural alterations of alpha-crystallin during its chaperone

action. Eur. J. Biochem. 258, 170 – 183.

60. Smulders, R. H., and de Jong, W. W. (1997) The hydrophobic probe

4,40-bis(1-anilino-8-naphthalene sulfonic acid) is specifically photo-

incorporated into the N-terminal domain of alpha B-crystallin. FEBS

Lett. 409, 101 – 104.

61. Liang, J. N., and Li, X. Y. (1991) Interaction and aggregation of lens

crystallins. Exp. Eye Res. 53, 61 – 66.

62. Stevans, A., and Augusteyn, R. C. (1997) Binding of 1-anilino-

naphthalene-8-sulfonic acid to alpha-crystallin. Eur. J. Biochem. 243,

792 – 797.

63. Burgio, M. R., Kim, C. J., Dow, C. C., and Koretz, J. F. (2000)

Correlation between the chaperone-like activity and aggregate size of

alpha-crystallin with increase in temperature. Biochem. Biophys. Res.

Commun. 268, 426 – 432.

64. Bova, M. P., McHaourab, H. S., Han, Y., and Fung, B. K. (2000)

Subunit exchange of small heat shock proteins. Analysis of oligomer

formation of alphaA-crystallin and Hsp27 by fluorescence resonance

energy transfer and site-directed truncations. J. Biol. Chem. 275,

1035 – 1042.

65. Bova, M. P., Ding, L. L., Horwitz, J., and Fung, B. K. (1997)

Subunit exchange of alphaA-crystallin. J. Biol. Chem. 272,

29511 – 29517.

66. Treweek, T. M, Rekas, A., Lindner, R. A., Walker, M. J.,

Aquilina, J. A., Robinson, C. V., Horwitz, J., Perng, M. D.,

Quinlan, R. A., and Carver, J. A. (2005) R120G alphaB-crystallin

promotes the unfolding of reduced alpha-lactalbumin and is inherently

unstable. FEBS J. 272, 711 – 724.

67. Bera, S., Thampi, P., Cho-W. J., and Abraham, E. C. (2002)

A positive charge preservation at position 116 of alpha A-crystallin

is critical for its structural and functional integrity. Biochemistry 41,

12421 – 12426.

68. Bhat, S. P. (2003) Crystallins, genes and cataract. Prog. Drug Res. 60,

205 – 262.

69. Kumar, P. A., Haseeb, A., Suryanarayana, P., Ehtesham, N. Z., and

Reddy, G. B. (2005) Elevated expression of alphaA- and alphaB-

crystallins in streptozotocin-induced diabetic rat. Arch. Biochem.

Biophys. 444, 77 – 83.

70. Horwitz, J., Bova, M., Huang, Q. L., Yaron, O., and Lowman, S.

(1998). Mutation of alpha B-crystallin: effects on chaperone-like

activity. Int. J. Biol. Macromol. 22, 263 – 269.

71. Smulders, R. H., Merck, K. B., Aendekerk, J., Horwitz, J.,

Takemoto, L., Slingsby, C., Bloemendal, H., and De Jong, W. W.

(1995) The mutation Asp69 ––4Ser affects the chaperone-like activity

of alpha A-crystallin. Eur. J. Biochem. 232, 834 – 838.

72. Shroff, N. P., Bera, S., Cherian-Shaw, M., and Abraham, E. C. (2001)

Substituted hydrophobic and hydrophilic residues at methionine-68

influence the chaperone-like function of alphaB-crystallin. Mol. Cell

Biochem. 220, 127 – 133.

73. Sreelakshmi, Y., and Sharma K. K. (2005) Recognition sequence 2

(residues 60–71) plays a role in oligomerization and exchange

dynamics of alphaB-crystallin. Biochemistry 44, 12245 – 12252.

PROPERTIES OF a-CRYSTALLIN 641