Biochimica et Biophysica Acta 1860 (2016) 333–343

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagen

Gamma crystallins of the human eye lens☆

Venkata Pulla Rao Vendra a, Ismail Khan b, Sushil Chandani c,
Anbukkarasi Muniyandi d, Dorairajan Balasubramanian b,⁎
a
Ophthalmic Molecular Genetics Section, National Eye Institute, Building 5635FL, Room 1S24, 5625 Fishers Lane, Rockville, MD 20852, United States
b
Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad 500034 Telangana, India
c
Plot 32, LIC Colony, W Marredpally, Secunderabad 500026, Telangana, India
d
Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Background: Protein crystallins co me in three types (α, β and γ) and are found predominantly in the eye, and
Received 17 April 2015 particularly in the lens, where they are packed into a compact, plastic, elastic, and transparent globule of proper
Received in revised form 8 June 2015 refractive power range that aids in focusing incoming light on to the retina. Of these, the γ-crystallins are found
Accepted 19 June 2015 largely in the nuclear region of the lens at very high concentrations (N 400 mg/ml). The connection between their
Available online 25 June 2015
structure and inter-molecular interactions and lens transparency is an issue of particular interest.
Scope of review: We review the origin and phylogeny of the gamma crystallins, their special structure involving
Keywords:
Human gamma crystallins
the use of Greek key supersecondary structural motif, and how they aid in offering the appropriate refractive
Greek key motif index gradient, intermolecular short range attractive interactions (aiding in packing them into a transparent
Structure–function correlation ball), the role that several of the constituent amino acid residues play in this process, the thermodynamic and
Cataractogenic mutations kinetic stability and how even single point mutations can upset this delicate balance and lead to intermolecular
Congenital cataract aggregation, forming light-scattering particles which compromise transparency. We cite several examples of this,
and illustrate this by cloning, expressing, isolating and comparing the properties of the mutant protein S39C of
human γS-crystallin (associated with congenital cataract-microcornea), with those of the wild type molecule.
In addition, we note that human γ-crystallins are also present in other parts of the eye (e.g., retina), where
their functions are yet to be understood.
Major conclusions: There are several ‘crucial’ residues in and around the Greek key motifs which are essential to
maintain the compact architecture of the crystallin molecules. We find that a mutation that replaces even one of
these residues can lead to reduction in solubility, formation of light-scattering particles and loss of transparency
in the molecular assembly.
General significance: Such a molecular understanding of the process helps us construct the continuum of
genotype–molecular structural phenotype–clinical (pathological) phenotype. This article is part of a Special
Issue entitled Crystallin Biochemistry in Health and Disease.
© 2016 Elsevier B.V. All rights reserved.

1. Gamma crystallins: phylogeny, presence in the lens, functions
Lens crystallins constitute about 90% of the total soluble protein con-
tent of the lens, and 35% of the total lens mass. They are composed of
two broad classes: α and βγ. The distribution of the three crystallins
in the lens is asymmetric and biphasic [1]. The central portion of the
lens is rich in β- and γ-crystallins, and the embryonic nuclear region,
from where the lens grows, is particularly rich in γ-crystallins. These
proteins are the earliest to be expressed as the lens is formed and
grows, and present in very high concentrations (N400 mg/ml in
☆ This article is part of a Special Issue entitled Crystallin Biochemistry in Health
and Disease.
⁎ Corresponding author.
E-mail addresses: pullaraovv@gmail.com (V.P.R. Vendra), ismailkhan.hcu@gmail.com
(I. Khan), sushilchandani@gmail.com (S. Chandani), anbu.blessings@gmail.com
(A. Muniyandi), dbala@lvpei.org (D. Balasubramanian).
mammalian eye lenses and N 1000 mg/ml in some fish lenses). They
are packed compactly in short range spatial order as in dense liquids
or glasses such that they aid transparency (no scattering) and the ap-
propriate refractive index gradient, high in the nuclear region and
lower in the cortical region of the lens. These two features are achieved
by the unique and stable structure and packing that they have adopted,
a classic example of structure dictating function.
Of the three crystallins found in the vertebrate lens (α, β and γ),
gamma crystallins are the smallest and simplest members. Each of
them has about 175 amino acids in its sequence, with a molecular
weight of about 21 kDa. Each of them is folded in a compact globular
manner and exists as a monomer. At least six different types of
γ-crystallins have been reported so far, namely γA–γF. In addition,
another one, which used to be called βS-crystallin, is now reclassified as
γS-crystallin, based on its similarity with the other γ-crystallins. The
human eye lens is known to contain γC, γD and γS crystallins (γE and

http://dx.doi.org/10.1016/j.bbagen.2015.06.007
0304-4165/© 2016 Elsevier B.V. All rights reserved.
334 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343

Table 1
Primary structures of human γC-, γD- and γS-crystallins.

An alignment of the amino acid sequences of human γC, γD and γS crystallins. Positions with invariant amino acids are shown in
bold. Serine 39 in γS crystallin is underscored. Colored stretches correspond to the four Greek key motifs.

γF- are pseudogenes in humans). While γC and γD are synthesized any, there. The putative role of lens crystallins, particularly γ-
during the embryonic stage, γS is synthesized post-natally and with de- crystallins, is an area that is drawing considerable current attention.
velopment their relative proportion decreases with concomitant
increase of α- and β-crystallins. 2. Section I: the Greek key motif: role in structure and function
Gamma crystallin is part of the βγ-family and known to have evolved
from primordial sources, namely archaeabacteria, and a description of The primary structural sequences of human γ-crystallins are shown
such microbial βγ-crystallins has been provided recently by Mishra in Table 1.
et al. [2]. The solution state structure of such a primordial βγ-crystallin A common protein structural folding pattern that has been pre-
from Archaea has been published by Barnwal et al. [3]. The native state served through the phylogeny is the so called Greek key motif, a
stability and related properties of the βγ-crystallins have just been super-secondary structural fold that was originally defined and named
reviewed by Serebryany and King (4). How these proteins have evolved by Richardson [8]. The Greek key motif involves a run of four β-sheet
over time in order to play a role in the vertebrate eye lens, and what structures folded in an antiparallel manner to generate a neatly folded
their functions are in and out of the lens have been comprehensively sum- compact domain, as shown in Fig. 1. Several archaeal proteins such as
marized recently by Slingsby, Wistow and Clark [5], and Wistow and Nitrollin or M-crystallin are folded using one such Greek key domain
Slingsby [6]. Given that these comprehensive reviews [2,4–6] have just while the eye lens βγ-crystallins are two-domain structures (with two
appeared, and that the present article is the lone one on γ-crystallins in Greek key motifs per domain), and the protein referred to as mammali-
this special issue, we first present here a ‘review of reviews’ on human an Absent in Melanoma 1 (AIM-1) uses as many as six such domains [2].
γ-crystallins, and follow it up with how mutations in them are associated Indeed, it is this folding using the Greek key motif that renders
with congenital cataracts in humans, an area of special interest to eye care the γ-crystallins with a variety of favorable features and properties
centers such as ours; our center alone treats as many as 1200 such pa-
tients each year. Congenital cataracts are largely genetic in origin (muta-
tions in over 45 genetic loci and over 38 genes in humans [7]), unlike
other forms of cataract where metabolic, environmental and post-
translational modifications can alter the structural and functional proper-
ties of the constituents of the lens; also a large fraction of them are Men-
delian in origin (one gene–one phenotype) so that a genotype–
phenotype correlation is easier. Further, since the detailed molecular
structures of several lens crystallins, particularly the βγ-crystallins are in-
creasingly being unraveled, we can attempt a genotype–molecular struc-
tural phenotype–clinical phenotype correlation.
We are interested here in congenital cataracts associated with
mutations in human γ-crystallins (as many as 30 are reported to date
in γC, γD and γS-crystallins). Since the detailed molecular structures
of these molecules have been solved, making such a genotype–structural
phenotype–clinical phenotype correlation appears to be a possibility.
We have divided this paper into three broad sections. In the first
section, we review the results of a significant number of papers in
the literature which have described the unique structural features of
γ-crystallins, namely, the Greek key motif, and summarize how folding
of the molecule using this motif offers these proteins the right set of
properties needed in the human lens, such as compactness, right
refractive properties, stability and intermolecular packing to generate
a transparent assembly.
In the second section, we show how single point mutations in their se-
quence can lead to perturbations in the structure leading to intermolecu-
lar aggregation and the consequent compromise in lens transparency. As
an example, we present our own ongoing research on the mutant S39C of Fig. 1. The Greek key motif. A: an artistic rendering of the motif; B: a schematic of the ar-
rangement of beta strands in one Greek key domain of the γ-crystallins; C: ribbon diagram
human γS-crystallin, associated with cataract and microcornea in a child. of γS crystallin with the motifs in blue, green, yellow and red, and D: Corey–Pauling–
In the third section, we briefly discuss the presence of crystallins in Koltun space-filling model rendering the same perspective as in Fig. 1C and E rotated
tissues outside the lens and some ongoing research on their role, if 90° for a view from the top.
 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343 335

suited for their role in the lens. The intramolecular packing involving
the coming together of the N-terminal and C-terminal domains
generates a compact monomeric globule of about 5 nm in size,
i.e., Stokes radius of 2.13 nm [9], and a low frictional ratio of about
1.21 (only slightly above the value of 1.12 for a perfect compact smooth
protein sphere), suggesting that they have low hydration and a low
propensity for sticky interactions with solvent and perhaps, with
other proteins [10]. These small globules, packed at such high concen-
trations in the lens (400–1000 mg/ml), are seen to exhibit short range
order which is necessary for transparency, particularly in a crowded
macromolecular environment as in the lens [11]. Interestingly, it has
been reported that while interactions between γ-crystallins are weakly
attractive, those between β-crystallins appear to be mildly repulsive [12].
Table 1 lists the sequences of the three γ-crystallins found in
humans. Human γC-crystallin, a protein of 174 residues, is folded in
four Greek key motifs, residues 2–40 forming the first motif, residues
41–83 forming the second (both in the N-terminal domain of the
molecule) and residues 88–128 making up the third and 129–171
making up the fourth and last Greek key fold. Residues 84–87 make
up the ‘linker region’ which, being a short one, brings the N-terminal
and C-terminal domains fold upon each other, thus making the mole-
cule a compact globule. Human γD-crystallin is folded in exactly the
same way as human γC-crystallin. In human γS-crystallin, shown in
Fig. 1, the sequence 6–44 folds into one Greek key motif, the residues
45–87 fold as the second, while the sequence 94–134 forms the third
and the stretch 135–177 adopts the last motif. Residues 88–93 form
the connecting peptide that allows the intramolecular coming together
of the N-terminal and C-terminal domains to produce a compact
globular molecule. (In contrast, the linker region plus N-terminal and
C-terminal extensions in the β-crystallins together lead to inter-
molecular, rather than intra-molecular, interactions generating dimers
and multimers [13,14] rather than monomers like in γ-crystallins.)
Also, while the βγ-crystallins fold using the Greek key motifs,
α-crystallin, which belongs to the small heat shock protein family,
does not. It is a multimeric protein with molecular weights as high
as 800 kDa. The structures and properties of some members of the β-
crystallin family, and those of α-crystallins are dealt with by other
contributors to this special issue.
The crystal structure of calf lens gamma-II crystallin was first pub-
lished by Blundell et al. [15] and Wistow et al. [16], and that of human

Fig. 2. Tyrosine corners and domain interface. Fig. 2A: The tyrosine corner domain interface in human γD-crystallin. In the N-terminal domain (left), the side chain hydroxyl group of Y63
forms a H-bond with the backbone oxygen of R59 (residue N-4). In the C-terminal domain, it is Y151 and M147. The inset shows only the N-terminal domain tyrosine corner; the three
intervening residues are in gray. Fig. 2B: Residues involved in stabilizing the domain interface in human γD-crystallin. Three hydrophobic residues from each domain are depicted in the
CPK structural format; the two polar residues of each domain are shown in the ball-and-stick format. The hydrogens have been stripped for clarity, and the carbon atoms in residues in the
N-terminal domain are colored green, while those in the C-terminal domain are in pink.
336 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343

γD-crystallin by Basak et al. [17]. (Also, the crystal structure of the C-
terminal domain of human γS-crystallin has been determined [18].)
The molecule is seen to pack in a highly symmetric manner, with each
of the two domains as tightly packed β-sheet sandwiches made of
Greek key motifs. As Chen et al. [12] point out, this template structure
is highly conserved, thanks to several key residues in the sequence
which play essential roles in the folding pattern, yet tolerates some var-
iations in the non-essential residues in the sequence. The relation be-
tween the role of the Greek key structure and the values and gradients
in the refractive index of lens proteins has been drawn attention to in
some recent papers [19,20].
The function of the lens is to focus the light coming through the cor-
nea into the retina. This would need not only the appropriate refractive
index values, but also a refractive index gradient which minimizes
spherical aberration. The lens should also be plastic (changeable in
shape) and elastic (return to original shape after pressure is released),
so as to accommodate its size and shape in a manner such that near ob-
jects as well as far objects should be focused on the retina. While the av-
erage refractive index of the nuclear region of the porcine lens is about
1.45, it decreases to about 1.36 as we move from the central to the cor-
tical region on the equatorial plane [21]. The macromolecular concen-
tration gradient (very high levels to gradually lower ones) that is seen
as we go from the nuclear to the cortical region of the live lens is in
what has been called opto-biological synchrony [22]. The refractive
index of a protein can be estimated based on the refractive index incre-
ment (dn/dc) values from the amino acid composition, using known
amino acid refractivity values of each constituent residue [23], and not
necessarily its actual sequence. It is noted that the dn/dc values of
double Greek key domains are consistently higher (0.2009 ml/g for
the Greek key motifs of crystallins, cf. 0.1899 for other human proteins
[19]). Likewise, Zhao et al. [20] had earlier argued that “one could spec-
ulate that this flexibility of the Greek key motif to accommodate differ-
ent residues lends itself well as a stable scaffold for lens proteins,
providing the opportunity of replacing residues with low dn/dc with
high ones”. It should also be noted that γ-crystallins have the highest
dn/dc values, namely 0.203 ml/g while β-crystallins have the lowest
(0.187 ml/g, and α-crystallins in between [24]). This is in keeping
with the fact that γ-crystallins are the most abundant proteins in the
core of the lens where the refractive index is the highest.
Table 1 above lists the amino acid sequences of the three human γ-
crystallins, namely γC, γD and γS. Note the remarkable sequence ho-
mology between the three, suggesting as well that they will all be folded
in much the same manner, which has proved to be the case.
The crystal structural analysis of γ-crystallins shows all nonpolar
residues in the molecule to be packed in the interior and polar ones
on the surface. Solution state studies also bear this out. This also
makes the molecule both thermodynamically and kinetically stable.
The intra- and inter-molecular packing features of the molecule (partic-
ularly human γC, γD and γS), and the roles played by various constitu-
ent residues have been dissected and reported in a series of about 30
papers by Jonathan King's group [25] and in about 12 papers by the
Pande group [26] using optical spectroscopic and thermodynamic

Table 2
Mutations reported in human γD-, γC- and γS-crystallins (taken with permission from Vendra et al. [62]).

S. no. Mutation Type of cataract seen Reference Comments

A: Mutations reported in human γD-crystallin

1 R14C Nuclear and perinuclear [45,46] Only surface change; extensive disulfide intermolecular bridges (see ref. 47)
2 R14S Coralliform [48] Slight change in hydrophobic content, opening up a phosphorylation site (no conformational work)
3 P24T Coralliform, cerulean, fasciculiform [49–55] Extensive conformational analysis done by many shows no change in sec/tert structure, minor
surface changes, drop in solubility (see refs. 56–60)
4 A36P Nuclear [61] Greek key compactness distorted, higher hydrophobic exposure upon mutation; solubility drops
(see ref. 62)
5 R37P Nuclear opacity [63] No data yet
6 R37S Birefringent crystals [64] Crystal structure reveals no changes (see ref. 65)
7 W43R Dominant congenital cataract [44] Minor tert struct. change; pI must be different, solubility not affected (see ref. 44)
8 M44V Blue dot opacity [66] No structural data yet
9 Y56X Nuclear [67] Large scale truncation of chain; three Greek key motifs lost
10 R58H Aculeiform [68] H bonding lost, but no other change. pI altered (see ref. 65)
11 G61C Coralliform [69,70] Mutation mainly affected the transition from native to intermediate state but not from intermediate
to unfolded form.
12 R77S Polar coronary cataract [71,72] No change seen in structure upon mutation. pI change? Solubility high (see ref. 62)
13 E107A Nuclear [73,74] Same as above. Solubility high. pI change is seen to lead to heteroaggregation with α-crystallin
(see ref. 62)
14 Y134X Not reported [75] No data, but Greek key 4 lost (see ref. 62)
15 R140X Nuclear [76] Greek key 4 lost; solubility lost; surface exposure extensive (see ref. 62)
16 W157X Nuclear [77] Greek key 4 lost; solubility lost; surface exposure extensive (see refs. 62,78)
17 G165fs Nuclear [79] High hydrophobic exposure; solubility lost (see ref. 62)

B: Mutations reported in human γC-crystallin

1 T5P Coppock-like cataract [68] No data
2 G62fs Zonular pulverulant [80] No data
3 C109X Nuclear cataract [81] Truncation and loss of Greek key
4 S119S Nuclear cataract [67] Polymorphism
5 W157X Nuclear cataract [82] Truncation mutant; data shows solubility loss, exposure of residues to surface (see refs. 62,78)
6 R168W Nuclear cataract [77,83] pI change, as in E107A? (see ref. 62)
7 R48H Nuclear cataract [84] Similar to the γD- mutant
8 G129C Nuclear cataract [85] Tertiary structure appears impaired

C: Mutations reported in human γS-crystallin

1 G18V Progressive cortical cataract [86] Extensive data shows no change in 2° or 3°, but compaction of GK 1 likely to alter (see refs. 87,88)
2 D26G Coppock cataract [61] Very little change in 2° or 3°, solubility high (see ref. 89)
3 S39C Microcornea-cataract [76] Surface hydrophobicity increases, causing the tendency to self aggregate (current study)
4 V42M Autosomal dominant cataract [90] Compact packing of Greek key is distorted (see refs. 91,92)
5 G57W Autosomal dominant pulverulent cataract [93] G highly conserved. Substitution by W partially disrupts the second Greek key motif.

Residue numbers in column 2 are as reported with cited references, regardless of whether methionine1 was counted or not. Note-2: Coralliform: round or elongated processes radiating
out of the center of the lens; cerulean: small bluish dots; fasciculiform: fibrous radiative strands emanating from the center; aculeiform: frosted lens; polar coronary: ring around a clear
central lens, slowly progressive cortical opacity; Coppock-like: bilateral progressive opacity of the embryonic nucleus, pulverulent (fine powderish); zonular/lamellar: affecting only cer-
tain layers between nucleus and cortex.
 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343
methods, and by NMR methods, notably by the groups of Gronenborn
[27], Martin [28] and others.
These studies have revealed several interesting properties of the
molecule. For example, γD-crystallin is highly stable, not denatured by
the conventional denaturing agent urea, and needs over 2.5 M of the
stronger denaturant guanidinum chloride [29]. In contrast, βB1, βA1,
βA3 and βB2 crystallins denature in urea [30,31]. The free energy of
denaturation of human γD-crystallin has been estimated to be about
8.9 kcal/mol [32], and of γS to be 10.5 ± 0.9 kcal/mol [33]; in compari-
son, that of αA-crystallin is 6.38 kcal/mol and αB 5.04 kcal/mol [34].
Similarly, the thermal denaturation temperature of γS is about 74.1 °C
and of γD is 83.8 °C [33], although it is pH dependent [35], while
those of β-crystallins are lower; e.g., that of βB2 is around 58 °C [36]
and βB1 about 67 °C [37]. It is also interesting to note that the stability
of the N-terminal domains of γ-D and γ-S crystallins are inherently
lower than those of their C-terminal domains [33]. Also, γ-crystallins
are kinetically stable, making them long-lived. The half-life of the initial
unfolding step of γD-crystallin is estimated to be around 19 years [38]!
3. Roles of some constituent amino acid residues in the structure
and stability; relation between structural changes and
lens malfunction
While it is true that the Greek key motif is adopted by a variety of
proteins with differing primary structural sequences [2], there are
some constituent residues that play an important role in maintaining
the architecture of human γ-crystallins. These have been studied in
great detail by a number of workers cited above.
The role played by the aromatic residues needs special mention.
Human γC-crystallin has a total of 22 aromatic residues (14Y, 4W and
4F), γD has 24 of them (14Y, 4W and 6F), while γS-has 27 of them
(14Y, 4W and 9F). Of these, the Y residues have a particularly important
role in the stability of the Greek key fold, by placing themselves in what
has been termed as the tyrosine corner [39], wherein a tyrosine residue
near the beginning or the end of an antiparallel β-strand hydrogen
bonds with a residue (often the Y-4 residue, or Δ4 Tyr corner) thus sta-
bilizing the β-barrel structure of the Greek key motif. For example, in
γC-crystallin we find two Δ4 Tyr type of homologous tyrosine corners,
one in N-terminal domain around Y63 (Y56, Y63 and W69) and another
one in the C-terminal domain around Y151 (Y144, Y151, W157), and
the same is true with γD, while the tyrosine corners in γS are (Y60,
Y67 and W73) and (Y150, Y157, W163).
Fig. 2A highlights two such tyrosine corners in human γD-crystallin,
those of Y63 and Y151. Yang et al. [40] have recently dissected the con-
tributions of the β-hairpin tyrosine corners to the stability and long life
of the γ-crystallins.
(Note: The amino acid residues in several publications are numbered
counting the N-terminal starting residue methionine as number 1
(e.g., P24T, R77S), while other publications discount met 1 and count
residues as (P23, R76). In order to avoid any confusion and maintain
uniformity, we number residues here counting the starting methionine
as met 1).
Besides tyr, the clustering of aromatic side chains within the interior
of the molecule is a feature that allows the γ-crystallins to take on a
compact shape with all nonpolar residues buried within. These aromatic
residues cluster into several structural elements. Besides the tyrosine
corners, six aromatic pairs (four tyr/tyr, one tyr/phe and one phe/phe)
are present in the β-hairpin sequences of the Greek key. The role of
these aromatic pairing to the stability of the molecule has been
highlighted by Kong and King [41]. The role that trp residues (W) play
is also worth commenting upon. In human γC and γD crystallins, all
the four trp residues are well buried at positions 43, 69, 131, 157. Of
the four trp residues, W69 and W157 are surrounded by aromatic
amino acids: Y56 and Y63 in the case of W69, and Y144 and Y151 sur-
round W157. Similarly, in γS, the trps are in positions 47, 73, 137 and
163. How these trp and tyr/cys clusters go to stabilize and protect γ-
337
crystallin has recently been studied [42,43]. Interestingly replacement
of even one of these, namely W43 (by R) is seen to lead to weakening
of the stability, loss of solubility and protein aggregation in human
γD-crystallin, as in the case of a human patient with cataract [44].
The two domains are covalently linked by a four/six residue-long
connecting peptide which allows them to interact non-covalently
through inter-domain contacts. This inter-domain interface is com-
posed of a cluster of six hydrophobic residues and two pairs of polar
peripheral interactions surrounding the hydrophobic cluster. These in-
terfacial residues of human γD-crystallin are illustrated in Fig. 2B. The
hydrophobic cluster consists of M44, F57, and I82 from the N-terminal
domain and V132, L145 and V170 from C-terminal domain. Peripheral
pair-wise interactions are between Q55/Q143 and R80/M147. How
such a hydrophobic inter-domain interface contributes to the folding
and stability of the molecule has been described [32].
Quite apart from the aromatics, several other residues (e.g., arginine,
cysteine, proline, serine) also contribute to the structure and stability of
human γ-crystallins. Replacement of even one such ‘critical’ residue by
mutation can lead to drastic changes in the protein's properties, which
translate into compromise in transparency of the lens and cataract.
4. Section II: mutations in human γ-crystallins and pathology
Table 2 lists as many as 30 such cataract-associated mutations in
human γC, γD and γS crystallins reported to date in human patients.
(It is interesting to note that most of these are single point mutations,
while some others are truncation mutations, one of them is a deletion
variant, and one a 5-base pair insertion.) They are all inherited (congen-
ital) cataracts with no post-translational or age-related modifications in
phenotype. These make it easy to clone the variant by site-directed mu-
tagenesis, isolate and purify the molecule and compare its structural
properties with those of the wild type protein, so as to understand
what the mutation has done to the properties of the protein.
The role played by the arginine residues (20 of them in human γC,
21 in γD, and 13 in γS) in maintaining the overall structural folding of
the native protein is exemplified in the Table, where we see that replac-
ing any of these by other amino acid residues (e.g., R15, R37, R59, R77S
and R140X in γD, R48 and R168 in γC) is associated with congenital
Fig. 3. Mutation leads to increased surface exposure. Panel A: An overlay of the wild type
(green; PDB code 2M3T) and S39C mutant (the red arrow points to the S residue). Panel
B: Y11, F16 and S39 residues in the wild type. Panel C: Y11, F16 and C39 in the mutant
model. Panel D: an overlay of B and C.
338 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343

cataracts. The γD mutants R37S and R59H are seen to be more prone to
crystallization than the wild type protein [65].
And the role played by cysteine residues (6 in γD, 8 in γC and 7 in
γS) has been studied in detail by the Pandes, who have, for example,
shown how when R15 is replaced by C in γD-, the resultant mutant
molecule undergoes intermolecular disulfide crosslinking, which
initiates protein aggregation [47]. Giblin et al. [94] have suggested that
S-thiolation within the molecule may act to delay the insolubilization
of the lens proteins.
Though the imino acid proline, both through its ring structure and
the lack of hydrogen-bonding ability, causes a break in the α-helical
and β-sheet runs, it seems to play a role in the folding of the
Greek key motif. For example, in γD-, replacing the residue P24 with T
(or other residues such as S or V), as reported in detail by the Pandes
[56–60], decreases the solubility of the protein by increasing hydropho-
bicity and restricting backbone flexibility, altering the temperature-
dependent solubility and the phase diagram. The roles of other amino
acids in the sequence, whose replacement is associated with lens mal-
function, are yet to be understood. We present some results obtained
with one such example involving the serine residue below.
5. Study of a serine mutant: S39C of human γS-crystallin
The role of serine residues in the molecule is yet to be clarified.
There are as many as 13 S residues in human γC, 17 in γD and 11 in
γS-crystallin. Yet, interestingly, Table 2 reports mutation in just one of
these so far, namely the single point mutation S39C in human γS-
crystallin, which is associated with micro-cornea and cataract [76].
S39 is conserved across all γ-crystallins (it is S35 in γC and γD), and is
thus thought to be important for the structure. To get an idea of the
change in local environment in the vicinity of residue 39, we built a mo-
lecular model of the C39 mutant using as template the γS-crystallin
structure provided by Kingsley et al. [95]. Fig. 3 shows the overlay of
the structures of wild type (WT) human γS and the S39C mutant. As
can be seen in Fig. 3C, the Y11 and, particularly, the F16 side chains
have moved outward and are somewhat more exposed in the mutant.
In order to get a molecular understanding of this cataract, namely,
how this replacement of S by C affects the structure of the mole-
cule, we have used site-directed mutagenesis of the mutant S39C
γS-crystallin molecule, cloned, expressed, purified and studied its
solution state properties, and compared them with those of native WT
molecule, and present some preliminary results here. The materials
and methods used for model building, cloning, over expression, isolation
and purification of the recombinant wild type and S39C mutant of
human γS-crystallin and the analysis of their structural and stability fea-
tures were done as described in our earlier publications [62,78,89,91].
We present the results here.
Turning to the results, we note from Fig. 4A that the gel elution profile
of the WT and the mutant S39C are essentially identical; the two mole-
cules elute at about the same volume, suggesting that the mutant is
quite soluble and has about the same size as the wild type. (There is a
hint of a higher molecular species eluting a little ahead (around
17.5 ml) of the main band in the mutant, but not in the wild type. We
are currently following this up in order to check whether it suggests mo-
lecular aggregation, e.g., dimerization, in the mutant protein.) Fig. 4B,
which describes the far-UV circular dichroism (CD) profiles of the two
proteins in the far-UV region (250–190 nm), shows that the secondary
structure of the two molecules is about the same. The characteristic neg-
ative band at 218 nm, positive band around 195 nm and a shoulder
around 206 nm are indicative of a high degree of β-sheet conformation

Fig. 4. S to C change at 39 does not affect the back bone conformation but slightly alters its tertiary structure. Panel A: Gel elution profile of wild type and mutant human γS-crystallin S39C.
If: fluorescence emission intensity in arbitrary units. λexc: 295 nm; the protein concentrations used were 0.825 mg/mL in 50 mM Tris-Cl (pH 7.3). Panel B: Far-UV CD spectra of wild-type
and mutant human γS-crystallin S39C. θ: elipticity in millidegrees. Protein concentration in each case was 0.150 mg/ml in 10 mM sodium phosphate buffer (pH 7.3). The cell path length
was 2 mm and all spectra were recorded at 37 °C, corrected for background buffer signal and each spectrum is an average of 3 independent runs. Panel C: Near UV CD spectra of wild type
and mutant human γS-crystallin S39C. θ: elipticity in millidegrees. Protein concentration in each case was 0.825 mg/ml in 50 mM Tris-Cl (pH 7.3). The cell path length was 10 mm and the
other conditions of measurement were the same as above. Panel D: Intrinsic fluorescence of wild type and mutant human γS-crystallin S39C. If: fluorescence emission intensity in arbitrary
units. λexc: 295 nm; The protein concentrations used were 10 μM (0.2 mg/mL) in 100 mM sodium phosphate buffer (pH 7.0). The cell path length was 3 mm, excitation and emission slits
2.5 nm and spectra were recorded at 37 °C.
 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343 339

Fig. 5. Panel A: Surface exposure of residues in the wild type and mutant human γS-crystallins S39C, monitored using bis-ANS as the extrinsic probe. λexc: 390 nm, If at 490 nm of the probe
was measured as a function of its increasing concentration. The cell path length was 3 mm, excitation and emission slits 2.5 nm, and spectra were recorded at 37 °C. Each point is an average
of 3 independent runs. Panel B: Aggregation tendencies of the wild type and mutant human γS-crystallin S39C, estimated using Nile Red as the extrinsic probe. λexc: 540 nm, If at 605 nm of
the probe was measured as a function of its increasing concentration. The cell path length was 3 mm, excitation and emission slits 10 nm, and spectra were recorded at 37 °C. Each point is
an average of 3 independent runs.

in the secondary structure of the protein chain, expected of the Greek key
fold. CD spectra in the near-UV region (340–250 nm), shown in Fig. 4C,
focusing on the micro-environmental region of the aromatic residues,
shows a slight difference between the two molecules, essentially in
their intensities. But such comparisons are better made and more infor-
mative, rather than near UV-CD, when one looks at the intrinsic fluores-
cence features of the aromatic residues, particularly trp, which has the
highest quantum yield of emission (with the wavelength of excitation
at 295 nm). When the microenvironment of the aromatic residue is non-
polar, the emission maximum occurs in the 320 nm region, and the emis-
sion quantum yield is rather low. But when the residue experiences a
more polar environment, its emission band is red-shifted and the inten-
sity of emission increases. Fig. 4D compares the emission profiles of the
two proteins; we find that while the band maximum is only slightly
red-shifted (by 1 nm) in the mutant compared to WT, and the band in-
tensity here has increased modestly, by about 10% compared to that of
the wild type, suggesting that the local environment around the once
buried aromatics has become somewhat more exposed.
Such changes in the surface polarity are better monitored using dyes
such as bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate) [96],
and 9-diethylamino-5H-benzo α-phenoxazine-5-(one) called Nile Red
[97] as extrinsic probes which, upon binding to exposed hydrophobic
surfaces display significant changes in their emission maxima and in-
tensity. Fig. 5A shows that when 140 μM of bis-ANS is titrated with
the proteins, its emission intensity is significantly higher (19 arbitrary
units) when bound to S39C than when bound to the WT (10 units),
suggesting that the mutant molecule has opened up a little more, thus
offering a greater surface for dye binding.
Does a more exposed surface allow the molecule to display a greater
tendency to make intermolecular interactions and thus aggregate? This
point can be checked by using the aggregation monitor dye Nile Red.
Fig. 5B compares the binding of this neutral dye Nile Red. Upon binding
to the wild type, it displays a broad band maximum centered around
651 nm with an intensity of about 0.65 arbitrary units, while upon bind-
ing to S39C, the band blue-shifted by 15 nm to 636 nm, and the intensity
doubled to over 1.3 units; this result too suggests that as the S residue is
replaced by the slightly bigger C, the tendency of the protein to offer a
somewhat more polar surface and promote intermolecular aggregation
becomes noticeable.
The temperature-dependent unfolding of the two proteins was next
studied by following the relative intensity ratios of fluorescence at
350 nm (representative of the red-shifted band maximum obtained
upon total unfolding of the molecule) and at 327 nm (representing
the native form). Fig. 6A shows that while the WT molecule starts to un-
fold after 60 °C, with a midpoint at around 74 °C, the mutant unfolds at a
much earlier temperature, well below 70 °C, with the midpoint around
59 °C, suggesting that the mutant has a lower thermal stability. Indeed,
the mutant starts scattering light already by about 50 °C during the runs,
hampering the accuracy of the experiment. Fig. 6B describes such time-
dependent increase in light scattering. While the wild type molecule

Fig. 6. Mutant has less thermal stability and is prone to aggregate faster. Panel A: Thermal denaturation of wild type and mutant S39C; λexc: 295 nm; The relative emission intensity of the
350 nm band was compared to that of the 327 nm band and monitored as a function of time. Protein concentration in each case was 0.065 mg/ml in 50 mM Tris-Cl (pH 7.3) and heated in
the range of 30–80 °C. The cell path length was 10 mm, excitation and emission slits 10 nm and spectra were recorded at 37 °C. Sample was allowed for 1 min prior to recording the spectra.
Panel B: Thermal aggregation of wild type and mutant at different temperatures. Light scattering was monitored at 600 nm light for 625 s at different temperatures (43.5, 53.5 and 54.0 °C).
A protein concentration of 0.260 mg/ml in 50 mM Tris buffer was used. The cell path length was 10 mm, excitation and emission slits 5 nm and spectra were recorded at 37 °C.
340 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343

Fig. 7. Legend: Mutant is less stable towards the chemical denaturant GuHCl and capable of forming an intermediate state during unfolding. Guanidine hydrochloride-induced denatur-
ation of wild type (curve A) and S39C (curve B) γS-crystallin. λexc: 295 nm; the relative emission intensity of the 350 nm band (of the denatured form) was compared to that of the 327 nm
band (of the native protein) and monitored as a function of denaturant concentration. The solid line indicates the fitted data and solid blocks stand for raw data. Protein concentration in
each sample was fixed at 0.2 mg/ml in 50 mM Tris buffer, 1 mM EDTA and 5 mM DTT. The cell path length was 3 mm, excitation and emission slits 5 nm, and spectra were recorded at 37 °C.

remains clear in solution at 54 °C for as long as 600 s, as monitored by
light scattering at 600 nm, the mutant aggregates to produce scattering
particles even when allowed to stand at 53.5 °C at 400 s; this is further
hastened to 300 s, when it is incubated at 54 °C.
We next studied the chemical denaturant-induced unfolding of the
mutant. The denaturant guanidine hydrochloride (GuHCl) was used to
unfold the protein and the relative fluorescence ratio (350 nm/
327 nm, representative of the ratio of unfolded/native forms of the
protein) was used as the assay. As Fig. 7A shows, the wild type molecule
displays a clear, sharp two-state transition from the native to the un-
folded form, with the midpoint of unfolding occurring at the concentra-
tion (Cm) of 2.8 M GuHCl. Data from this equilibrium native to the
unfolded two-state (N–U) curve were fitted to the two-state transition
model of Greene and Pace [98], yielding a free energy (ΔG0N–U) value of
9.145 ± 0.30 kcal/mol. In contrast, the denaturation curve (Fig. 7B) for
the mutant displays a multiple step process; the first transition already
occurs well before 1.5 M GuHCl, and the second at 2.8 M GuHCl, just as
with the WT molecule. When these data were analyzed using the three-
state model, proposed by Clark et al. [99], we obtained a Cm value of
0.73 M GuHCl and a (ΔG0N–I) value of 4.32 ± 0.26 kcal/mol, and a Cm
value of 2.8 M GuHCl for the second transition (I–U) with a ΔG0I–U
value of 4.88 ± 0.20 kcal/mol (see Table 3). The behavior of S39C is
thus reminiscent of some other mutants that we had described earlier,
e.g., V42M human γS- and A36P γD-crystallins.
We next examined the changes that the mutation brings about in
our molecular model which was built following protocols described in
our earlier reports [62,78,89,91] using the Swiss-Model workspace
[100] and refined by molecular dynamics procedures in Gromacs 4.5.5
[101]. Fig. 3, shown above, suggests that in the wild type molecule, res-
idues Y11 and F16 are in close contact, and S39 is within interacting dis-
tance. When it is replaced by C in the mutant, both Y11 and F16 move
outward in order to accommodate the bulkier sulfur atom in C39. Fur-
ther analysis shows that there is a change in the aromatic–aromatic
interactions between the two residues, with the centroid–centroid dis-
tance between Y11 and F16 increasing to 6.2 Å in the mutant when
compared to 5.5 Å in the wild type γS-crystallin. Both S39 and C39
form main chain–main chain H-bonds with the N of Y11. The side
chain oxygen of S39 forms H-bonds with the main chain N atom of
F16 and the O of D13. The larger S atom in the mutant forms these
two H-bonds and, in addition, forms H-bonds with the N atom of N15
and the O of P68. The S atom is also involved in the aromatic–sulfur in-
teraction with the F16, the centroid-sulfur atom distance being 4.5 Å.
However, the larger bulk of the S atom pushes out F16, which also
leads to a disruption, in the mutant. Cation-pi interaction seen between
F16 and K41 (distance 3.7 Å) in the wild type protein. Fig. 8 gives a com-
parison in our model of the inter-atom interactions between Y11, F16
and S39 (or C39 in the mutant), while Table 4 estimates the distances.
Note the small but noticeable increase in the distance between the
backbone C atoms of residues 16 (F16) and 39 (S39 or C39) (from
6.60 to 7.34 Å) and also of the N atoms (5.42 to 5.991 Å), while there
is some shrinkage between those of Y11 and S/C39.
It is also worth pointing out that the mutation involves the replace-
ment of serine (with a relative polarity ranking of 14 (based on the rel-
ative hydrophobicity scale), an average percentage of about 8% burial in
proteins, a van der Waals volume of 73 Å3, and accessible surface area of

Table 3
Equilibrium unfolding parameters for wild type human γS-crystallin and its S39C mutant.

Protein Equilibrium transition 1 Equilibrium transition 2

Cm Apparent ΔG0 P value Cm Apparent ΔG0 P value

Wild type 2.8 M 9.15 ± 0.3 0.001

[ΔG0N–U]
Fig. 8. Mutation alters the inter-atomic distances: H-bond interactions around residue 39
S39C 0.73 M 4.32 ± 0.26 0.002 2.8 M 4.88 ± 0.20 0.002
in the wild type and S39C mutant of γ-S crystallin. (A) Wild type protein, with the side
[ΔG0N–I] [ΔG0I–U]
chain oxygen of S39 shown as a Corey–Pauling–Koltun rendering and (B) the S39C
[Cm] = transition midpoint in the units of M GuHCl; [ΔG0] = free energy of unfolding in mutant, with the sulfur atom rendered in CPK. Arrows point to hydrogen bonds between
the absence of GuHCl in units of kcal/mol−1; N–U refers to native to unfolded transition, neighboring residues and residue 39. Note that the H-bond pattern between these
N–I native to intermediate and I–U intermediate to unfolded state. residues is not significantly altered in the mutant.
 V.P.R. Vendra et al. / Biochimica et Biophysica Acta 1860 (2016) 333–343
Table 4
Alterations in the inter-atom distances upon mutation.
Distance, 2M3T, wild S39C mutant 2M3T, wild
in Å Type C–C, C–C type N–N
distance, Å distance, Å distance, Å
Y11–F16 6.12 6.14 6.57
Y11–S/C39 5.50 4.82 5.24
F16–S/C39 6.60 7.34 5.42
C–C: backbone carbon to carbon distance, and N–N backbone nitrogen atoms distance,
both in Å units. 2M3T refers to the PDB code for human γS-crystallin.
80 Å2 by Cys (relative polarity ranking 7, average percentage burial
value of 3%, van der Waals volume of 86 Å3 and accessible surface area
of 104 Å2) [102]; in other words, even this rather minor alteration
weakens the stability of the molecule and makes it a little more hydro-
phobic on the surface, causing the tendency to self-aggregate and
generate light scattering particles which, in the lens, can lead to the clin-
ical phenotype of lens opacification and cataract. We had earlier shown
a similar genotype–molecular phenotype correlation with the muta-
tions V42M and D26G in human γS-crystallin [89,91], and Bharat et al.
[92] have confirmed this in greater detail in V42M, using NMR spectros-
copy; others had shown similar effects with the mutant G18V of the
same molecule [87,88], and DiMauro et al. [103] have shown that
even a subtle alteration such as acetylation of gly1 and lys 2 in human
γD-crystallin promotes aggregation of the otherwise highly soluble
molecule. We had earlier argued that the structural integrity of the
Greek key fold seen in lens crystallins appears essential for lens trans-
parency, and a minor distortion in even one of the Greek key folds in
lens crystallins leads to compromise in lens transparency [62]. The pre-
liminary results we have described above with S39C appear to be in ac-
cord with this argument. We still need to investigate the ultimate status
of the C residues in the self-aggregation.
6. Section III: gamma crystallins outside the lens
Having looked at the presence of, and the role played by γ-
crystallins in the lens, we now move to their presence outside the
lens, in other tissues of the eye. While there are many reports for the
non-lenticular presence and functions of α-crystallins, and also some
members of the β-crystallin family, particularly in the retina, the roles
of γ-crystallins are just getting to be understood [104,105]. Based on
the idea that γS-crystallin is an outlier of the γ family, with some char-
acteristics that appear more likely to retain non-lens function, Sinha
et al. [106] looked for the Crygs gene in the mouse eye and found the
γS mRNA to be present in the retina as well as the cornea, though not
in the iris. They wondered whether γS may have a role as a stress-
modulator in the tissues of the eye. Most of these studies so far have
concentrated on the murine eye, where they are suggested to play the
role of stress-modulators during the development of the eye. Since
then, several papers have appeared which have tried to follow up on
this idea [107,108]. However, all of these studies, while mentioning
βγ-crystallins, focus on the roles of specific β-crystallins such as βA3/
A1 [108], or βB2 [105], and these too on rodent eyes, rather than
humans. And when talking about γ-crystallins, reference is made invari-
ably to the mixture rather than a single member such as γS, γD or γC.
The role of individual members of human γ-crystallins is thus an issue
worth researching on. This is particularly worthwhile since we find,
while working with clinical colleagues in the pediatric ophthalmic sec-
tion at our institute, occasional cases of children diagnosed with both
congenital cataract and persistent fetal vasculature (PFV, wherein the
hyaloid artery involved in nourishing the growth of the eye through
vasculo- and angiogenesis does not regress from the anterior portion
of the eye at the appropriate juncture, thus leaving the vasculature to
persist and block vision). Zhang et al. [109] had seen the expression of
γ-crystallins in the hyaloid tissue of an infant with microphthalmia
and PFV. Ongoing genetic analysis in one such patient in our center,
341
who has cataract, PFV and microcornea, has revealed a single point mu-
tation in human γC crystallin (R48H), but it is not clear what role the
S39C mutant mutation plays in each one of these conditions. Work of this kind is ex-
N–N pected to give us an indication of the role that γ-crystallins play in non-
distance, Å lenticular parts of the eye.
6.98
4.74
Conflict of interest
5.99
None of the authors has any financial disclosures to make.
Acknowledgments
This work was funded by the Hyderabad Eye Research Foundation.
AM thanks the Science Academies of India for a summer research
fellowship. We thank Drs. Yogendra Sharma and Rajeev Raman of the
Centre for Cellular and Molecular Biology, Hyderabad for the use of
the circular dichroigraph, and for helpful comments. Also, owing to
the page and reference number limitations, we could not cite all rele-
vant original research articles and thereby apologize to the authors
whose publications are not cited.
VPRV and DB conceived the idea, VPRV and AM did much of the ex-
perimental work, VPRV and DB analyzed and interpreted the data and
wrote the manuscript, IK participated in the analysis, modified the man-
uscript, and checked all the references, SC did the protein modeling
work, helped analyze the data, and modified the manuscript. All the au-
thors read and cleared the final manuscript.
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