Figure 2.

1: IPTG induction of A- and B-crystallin

Lanes 1 & 2: Induction A-crystallin before and after addition of 1mM IPTG. Lanes 3
& 4: Induction B-crystallin before and after addition of 1mM IPTG. Lane 5:
Molecular weight marker.

1 2 3 4 5
200 kDa
116 & 97 kDa
66 kDa

45 kDa

31 kDa

21 kDa

14 kDa
Figure 2.2: Purification profiles of recombinant A- and B-crystallins.
panel A & B: elution profile on DEAE-sephacel (ion-exchange). panel C & D: elution
profile on sephacryl-S300 (gel filteration). Peaks corresponding to A- and B-
crystallin are indicated by arrows.
Figure 2.3: Purification of A- and B-crystallin.
Panel A & B: Fractions of A-crystallin from DEAE-sephacel (ion-exchange) and
sephacryl-S300 (gel filtration) respectively. Panel C & D: Fractions of B-crystallin
from DEAE-Sephacel (ion-exchange) and Sephacryl-S300 (gel filtration) respectively.
Fractions corresponding to the A- and B-crystallin (subunit mass - 20 kDa) is
indicated by arrows.

A B

C D
Figure 2.4: Sephacryl-S300 (gel filteration) profile of the goat eye lens homogenate

1.2

1.0
H-crystallin L-crystallin
0.8
A280nm

-crystallin -crystallin
0.6

0.4

0.2

0.0
0 20 40 60 80 100 120 140 160
Volume (ml)

Figure 2.5: Purified crystallins.

Lane 1: A- , Lane 2: B-, Lane 3: goat L-crystallin and Lane 4: molecular weight
marker

1 2 3 4
200 kDa
116 & 97 kDa

66 kDa

45 kDa

31 kDa

21 kDa

14 kDa
Table 2.1: Client proteins and assay conditions employed for assessing the
chaperone-like activity (CLA) of -crystallin variants.
S. No.

Client protein

Assay conditions
(mg/ml)Chaperone

1 Goat βL-crystallin 0.05 Ratio (w/w)Client/ Chaperone

4.0 Heat - aggregation at 60oC,
(0.2 mg/ml) 50mM sodium phosphate buffer
pH 7.2, containing 100 mM
NaCl
2 Goat γ-crystallin 0.02 12.5 Heat - aggregation at 60oC,
(0.25 mg/ml) 50mM sodium phosphate buffer
pH 7.2, containing 100 mM
NaCl
3 Carbonic anhydrase 0.2 1.0 Heat - aggregation at 60oC,
(0.2 mg/ml) 50mM sodium phosphate buffer
pH 7.2, containing 100 mM
NaCl
4 Citrate synthase 0.05 1.0 Heat - aggregation at 60oC,
(0.05 mg/ml) 40mM HEPES-KOH buffer pH
7.9
5 Citrate synthase 0.05 1.0 Heat - aggregation at 45oC,
(0.05 mg/ml) 40mM HEPES-KOH buffer pH
7.9
6 Insulin 0.5 0.8 DTT - aggregation at 37oC,
(0.4 mg/ml) 50mM sodium phosphate buffer
pH 7.2, containing 100 mM
NaCl
7 Goat γ-crystallin 0.15 1.6 UV - aggregation at 25 oC,
(0.25 mg/ml) 50mM sodium phosphate buffer
pH 7.2
8 Glucose-6 0.1 200 Heat - inactivation at 42oC,
phosphate 100 mM Tris-cl, pH 7.0
dehydrogenase
(0.5 U/ml)
Figure 2.6: Formation of recombinant -crystallin heteropolymers.
Immunoprecipitation (IP) followed by Western blotting (WB) of -crystallin
heteropolymers with A to B 3:1 & 1:3 ratio were done using A or B specific
antibodies.

3:1 1:3 3:1 1:3

29 kDa

20 kDa

IP : A A B B
WB : B B A A
 Figure 2.7: Chaperone-like activity (CLA) of -crystallin variants.
Panel A: Representative graph of chaperone activity of -crystallin variants in
suppressing heat-induced aggregation of L-crystallin at 60oC. Panel B: Relative
chaperone activity (percentage protection) was determined considering aggregation of
L-crystallin in the absence of -crystallins as 100%. Data are mean ± SD (n=4).
Variations in superscripts indicate significance (P<0.05) of mean differences between
variants.

1 : L A c
0.8 2 : 3:1 80
3 : A 7
4 : 1:3 6
5 : B 5
0.6 6 : 1:1 60

% Protection
4 d
7 : L
A 360nm

a
3
0.4 40
2
b
0.2 20
1

0.0
0
0 10 20 30 40 50 A B L 3:1
Time (min)

1 : L A c
0.8 2 : 3:1
B
80
3 : A 7
4 : 1:3 6
5 : B 5
0.6 6 : 1:1 60 d
% Protection

4
7 : L a
3
0.4 e
40
2 be
b
0.2 20
1

0.0
0
0 10 20 30 40 50 A B L 3:1 1:3 1:1
Time (min)
 Figure 2.8: Relative chaperone activity of -crystallin variants as assessed by
suppression of heat-induced aggregation of citrate synthase at 60 oC (Panel A) and 45oC
(Panel B). Increased chaperone activity upon preheat treatment with heat-induced
aggregation of citrate synthase assay 45oC (Panel C). Percentage increase in chaperone
activity in Panel C was determined considering the activity of unheated -crystallin as
100%. Data are mean ± SD (n=4). Variations in superscripts indicate significance
(P<0.05) of mean differences between variants.

100 100 100

100
A A AB
d

80 a a a
80 a a a 80
80 a
a a
ac c
% Protection

% Protection
% Protection

Protection
60 60 d d 60
60 ac d

c c b c
40 40 40
40 b

b b b
20 20 20
20

0 0 00
A A
B BL L
3:1 3:11:3 1:3 1:1 1:1 A
A B
B LL 3:13:1 1:31:3 1:11:1

100 60
d B C
a
80 50

ac c
40 b
60 ac
% increase

b
30
b
40 b
20

20
10

0 0
A B L 3:1 1:3 1:1
A L 3:1
 Figure 2.9: Relative chaperone activity of -crystallin variants with heat-induced
aggregation of γ-crystallin at 60oC (Panel A) and UV-induced aggregation of γ-
crystallin at 25oC (Panel B). Data are mean ± SD (n=4). Variations in superscripts
indicate significance (P<0.05) of mean differences between variants.

8
0 5
0
a A
a a
ac 4
0
6
0 c
a
% Protection

% Protection
3
0
4
0
d
bd 2
0
b b
2
0
1
0

0 0

A 
B 
L 3
:1 1
:3 1
:1 
A 
B 
L 3
:1
5
0
A B
a a
ac 4
0
c
a
% Protection

3
0
b
d
bd 2
0
b b
b
1
0

0

B 
L 3
:1 1
:3 1
:1 
A 
B 
L 3:1 1
:3 1
:1
Figure 2.10: The chaperone activity of -crystallin variants as assessed by the
suppression of heat-induced aggregation of carbonic anhydrase at 60oC. Data are mean
± SD (n=4). Variations in superscripts indicate significance (P<0.05) of mean
differences between variants.

100 a
a
80
% Protection

60

b
40

c
20
d d

0
A B L 3:1 1:3 1:1
Figure 2.11: Relative chaperone activity of -crystallin variants with DTT-induced
aggregation of insulin at 37oC. Data are mean ± SD (n=4). Variations in superscripts
indicate significance (P<0.05) of mean differences between variants.

60 b
b
50
% Protection

40 a
a a
a
30

20

10

0
A B L 3:1 1:3 1:1
Figure 2.12: The chaperone activity of -crystallins variants as assessed by the
suppression of heat-induced inactivation of glucose-6-phosphate dehydrogenase
(G6PD) at 42oC. G6PD assay was performed by measuring the increase in absorbance at
340nm due to the reduction of NADP. Data represent percentage protection and are
mean ± SD (n=4).

50

40
% Residual activity

30

20

10

0
A B L 3:1 1:3 1:1
Figure 2.13: Far-UV CD profile (secondary structure) of -crystallin variants

4e+5

2e+5

0 L
Molar Ellipticity
A
-2e+5

-4e+5 B

-6e+5

-8e+5 1:3 1:1

3:1
-1e+6
200 210 220 230 240 250
Wavelength (nm)

Table 2.2: Percentage distribution of secondary-structural elements of unheated

(native) and pre-heated -crystallin variants.
Secondary-structural content among native and pre-heated -crystallin variants was not
significant (P>0.05) by Mann–Whitney test. Significance in difference (P<0.05) is
indicated by (∗) by comparing before and after pre-heat treatment. Results are
means±SD (n=4)

Sample -Helix β-sheet β-turn Random coil

native pre-heated native pre-heated native pre-heated native pre-heated
A 25.1 35.6 38.7 43.7 11.5 8.4 24.7 12.3
± 3.0 ± 4.2 * ± 6.5 ± 4.2 ± 4.5 ± 0.4 ± 1.1 ± 8.1
B 21.5 21.0 28.5 34.2 21.5 22.2 28.4 22.1
± 3.0 ± 0.6 ± 5.0 ± 3.8 ± 3.5 ± 3.8 ± 1.3 ± 7.9
L 27.3 31.8 34.5 37.1 17.2 12.0 21.0 19.1
± 2.0 ± 6.8 ± 9.5 ± 5.3 ± 7.3 ± 6.2 ± 2.1 ± 5.0
3:1 23.4 30.5 32.3 33.3 16.3 15.1 28.0 21.1
± 4.0 ± 6.6 ± 7.0 ± 3.5 ± 3.5 ± 7.6 ± 0.8 ± 6.6
1:3 23.2 22.2 30.7 37.2 20.6 16.0 25.5 24.6
± 3.5 ± 0.8 ± 2.0 ± 5.7 ± 3.4 ± 2.5 ± 2.0 ± 4.5
1:1 24.0 30.6 30.3 40.0 19.0 10.7 26.7 18.7
± 3.2 ± 7.8 ± 5.5 ± 2.2 * ± 3.6 ± 2.7 * ± 1.5 ± 5.6 *
Figure 2.14: Tryptophan fluorescence of -crystallin variants.

280 RT
F lu o re s c e n c e in te n s ity

180
6 1 : A
160 2 : L
5 3 : 1:1
140
4 : 3:1
4 5 : 1:3
120
6 : B
3
100 2
80
1
60

40

20
0
300 320 340 360 380 400
Wavelength (nm)
 Figure 2.15: Near-UV CD profile (tertiary structure) of panel A: native -crystallin
variants and panel B: comparative profile -crystallin variants of preheated which have
shown an alteration in tertiary structure upon preheating.

4000
6000 6 A 4
2
4000 5 2000

Molar Ellipticity
Molar Ellipticity
4
2000
0
3
0 3
2
-2000
-2000 1-A
1:A
2-3:1
1 2:pr
-4000 3-1:1
-4000 3:3:1
4-L 1 4:pr
5-1:3
-6000
6-B -6000
260 280 300 320 340 260 280 300 320

Wavelength(nm) Wavelength(nm)

4000
6 A 4 B
2
5 2000
Molar Ellipticity

4
0
3
3
2
-2000
1- A
1:A
2- 3:1
1 2:prHt A
3- 1:1
-4000 3:3:1
4- L 1 4:prHt3:1
5- 1:3
6- B -6000
260 280 300 320 340 260 280 300 320 340

Wavelength(nm) Wavelength(nm)