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Figure 2.1: IPTG induction of A- and B-crystallinLanes 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

21 kDa

31 kDa

45 kDa

66 kDa

116 & 97 kDa

200 kDa

14 kDa

2 3 4 51

21 kDa

31 kDa

45 kDa

66 kDa

116 & 97 kDa

200 kDa

14 kDa

2 3 4 5

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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.

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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-crystallinfromDEAE-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 BAA BB

C DCC DD

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

Figure 2.5: Purified crystallins.

Lane 1: A- ,Lane 2: B-,Lane 3: goat L-crystallin andLane 4: molecular weightmarker

Volume (ml)

0 20 40 60 80 100 120 140 160

A280nm

0.0

0.2

0.4

0.6

0.8

1.0

1.2

-crystallin

H-crystallin L-crystallin

-crystallin

116 & 97 kDa

21 kDa

31 kDa

45 kDa

66 kDa

200 kDa

14 kDa

1 2 3 4

116 & 97 kDa

21 kDa

31 kDa

45 kDa

66 kDa

200 kDa

14 kDa

1 2 3 4

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Table 2.1: Client proteins and assay conditions employed for assessing the

chaperone-like activity (CLA) of -crystallin variants.

S.

No.

Clientpro

tein

Chaperone

(mg/ml)

Client/Chaper

one

Ratio(w/w)

Assaycondit

ions

1 Goat L-crystallin

(0.2 mg/ml)

0.05 4.0 Heat - aggregation at 60oC,

50mM sodium phosphate bufferpH 7.2, containing 100 mMNaCl

2 Goat -crystallin(0.25 mg/ml)

0.02 12.5 Heat - aggregation at 60oC,50mM sodium phosphate buffer

pH 7.2, containing 100 mM

NaCl

3 Carbonic anhydrase

(0.2 mg/ml)

0.2 1.0 Heat - aggregation at 60oC,

50mM sodium phosphate buffer

pH 7.2, containing 100 mMNaCl

4 Citrate synthase(0.05 mg/ml)

0.05 1.0 Heat - aggregation at 60oC,40mM HEPES-KOH buffer pH

7.9

5 Citrate synthase

(0.05 mg/ml)

0.05 1.0 Heat - aggregation at 45oC,

40mM HEPES-KOH buffer pH

7.9

6 Insulin

(0.4 mg/ml)

0.5 0.8 DTT - aggregation at 37oC,

50mM sodium phosphate buffer

pH 7.2, containing 100 mMNaCl

7 Goat -crystallin

(0.25 mg/ml)

0.15 1.6 UV - aggregation at 25 oC,

50mM sodium phosphate bufferpH 7.2

8 Glucose-6

phosphate

dehydrogenase(0.5 U/ml)

0.1 200 Heat - inactivation at 42oC,

100 mM Tris-cl, pH 7.0

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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 specificantibodies.

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

29 kDa

20 kDa

IP : A A B BWB : B B A A

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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: Relativechaperone 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

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Figure 2.8: Relative chaperone activity of -crystallin variants as assessed bysuppression 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 inPanel Cwas determined considering the activity of unheated -crystallin as100%. Data are mean SD (n=4). Variations in superscripts indicate significance

(P

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Figure 2.9: Relative chaperone activity of -crystallin variants with heat-inducedaggregation of -crystallin at 60oC ( Panel A) and UV-induced aggregation of -crystallin at 25oC (Panel B). Data are mean SD (n=4). Variations in superscripts

indicate significance (P

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Figure 2.10: The chaperone activity of -crystallin variants as assessed by thesuppression of heat-induced aggregation of carbonic anhydrase at 60oC. Data are mean SD (n=4). Variations in superscripts indicate significance (P

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Figure 2.11: Relative chaperone activity of -crystallin variants with DTT-inducedaggregation of insulin at 37oC. Data are mean SD (n=4). Variations in superscriptsindicate significance (P

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Figure 2.12: The chaperone activity of -crystallins variants as assessed by thesuppression 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).

%R

esidualactivity

0

10

20

30

40

50

A B L 3:1 1:3 1:1

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Figure 2.13: Far-UV CD profile (secondary structure) of -crystallin variants

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 wasnot significant (P>0.05) by MannWhitney test. Significance in difference (P

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Figure 2.14: Tryptophan fluorescence of -crystallin variants.

Wavelength (nm)

300 320 340 360 380 400

Flu

orescence

intensity

0

20

40

60

80

100

120

140

160

180

4

1

2

3

5

6 1 : A2 : L3 : 1:14 : 3:1

5 : 1:3

6 : B

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Figure 2.15: Near-UV CD profile (tertiary structure) ofpanel A: native -crystallin

variants andpanel B: comparative profile -crystallin variants of preheated whichhave shown an alteration in tertiary structure upon preheating.

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

1

2

3

4

5

6

1 - A2 - 3 : 13 - 1 : 1

4 - L5 - 1 : 36 - B

A

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

1

2

3

4

1 : A

2 : p r H tA3 : 3 : 14 : p r H t 3 : 1

B

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

1

2

3

4

5

6

1 - A2 - 3 : 13 - 1 : 1

4 - L5 - 1 : 36 - B

A

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

1

2

3

4

1 : A

2 : p r H tA3 : 3 : 14 : p r H t 3 : 1

B

W a v e le n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

1

2

3

4

5

6

1 - A2 - 3 : 13 - 1 : 14 - L5 - 1 : 36 - B

A

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

1

2

3

4

1 : A

2 : p r H tA3 : 3 : 1

4 : p r H t 3 : 1

B

W a v e le n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

1

2

3

4

5

6

1 - A2 - 3 : 13 - 1 : 14 - L5 - 1 : 36 - B

A

W a v e l e n g t h ( n m )

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

MolarEllipticity

- 6 0 0 0

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

1

2

3

4

1 : A

2 : p r H tA3 : 3 : 1

4 : p r H t 3 : 1

B