stability chapter iv figures

8/14/2019 Stability Chapter IV Figures

1/14

Figure 5.1: Differential scanning calorimetric (DSC) profiles of -crystallinvariants. Thermal unfolding endotherms of -crystallin variants scanned from 10-100oC at a scan rate of 60oC /h. Continuous line represents experimental curve and

dashed line is the best fit of the experimental data to a two-state transition model using

Microcal Origin software. Arrows indicate the temperature at which the B and 1:3 gotprecipitated.

10 20 30 40 50 60 70 80 90

0

2

4

6

8

A

Cp(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

B

10 20 30 40 50 60 70 80 90

0

2

4

6

8

L

Cp

(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

3:1

10 20 30 40 50 60 70 80 90

0

2

4

6

8

A

Cp(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

B

10 20 30 40 50 60 70 80 90

0

2

4

6

8

A

Cp(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

B

10 20 30 40 50 60 70 80 90

0

2

4

6

8

L

Cp

(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

3:1

10 20 30 40 50 60 70 80 90

0

2

4

6

8

L

Cp

(kcal/mole/oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

3:1

10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

1:3

Cp(kcal/m

ole/oC)

Temperature (oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

1:1

Temperature (oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

1:3

Cp(kcal/m

ole/oC)

Temperature (oC)

10 20 30 40 50 60 70 80 90

0

2

4

6

8

1:1

Temperature (oC)


2/14

Table 5.1: Thermodynamic data from DSC profiles derived using the MicroCal

Origin software. Data are meanSE (n=3).

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

Scan rate at 30/hr

TmoC 58.950.12

55.170.24

62.100.12

59.720.34

58.310.12

62.070.41

H

(kcal.mol-1)45.080.18

62.950.40

44.200.59

80.700.63

72.100.20

70.311.24

Scan rate at 60/hr

TmoC 58.99

0.22

56.12

0.21

62.98

0.12

61.01

0.12

59.84

0.17

62.20

0.12

H

(kcal.mol-1)

49.48

0.23

64.25

0.18

61.59

1.30

64.58

0.30

60.56

0.27

94.33

1.98

Scan rate at 90/hr

TmoC 60.18

0.14

56.86

0.11

62.90

0.21

61.21

0.03

59.16

0.03

62.51

0.31

H

(kcal.mol-1)

45.22

0.30

67.37

0.13

62.70

0.13

73.75

0.42

51.78

0.37

69.29

1.67


3/14

Figure 5.2:Size-exclusion chromatography (SEC) of -crystallin variants. Elutionprofiles of normal (continuous line) and preheated (dashed line) -crystallin variants

on a TSKG4000SWXL gel filtration HPLC column. Elution positions of standardmolecular weight markers, thyroglobulin (650 kDa; position-1), ovalbumin (150 kDa;

position-2) and BSA (67 kDa; position-3) are indicated on the top.

Time(min)

0 5 10 15 20

A280nm

normal

preheated

1 2 3

A

L

3 :1

1 :3

1 :1


4/14

Figure 5.3: Representative DLS profile of A-crystallin.

Table 5.2: Hydrodynamic radii assessed by DLS analysis on OmniSize 3.0 software

provided by the DLS instrument (Viscotek 810). Data are meanSD (n=3)

Sample Hydrodynamic Radii Rh (nm)

unheated at 25oC preheated at 45oC preheated at 60oC

A 10.05 1.68 7.3 0.27 8.9 1.44 B 8.55 1.05 8.3 0.89 precipitated L 9.02 1.84 9.4 1.75 9.8 1.343:1 9.12 1.31 7.7 1.08 9.2 0.6

1:3 6.5 0.45 7.9 0.50 precipitated

1:1 8.23 1.80 9.9 1.00 10.7 1.2


5/14

Figure 5.4: Representative tryptophan fluorescence profiles ofpanel A: A andpanel

B: 3:1 ( A: B), as a function of GdmCl.

Wavelength (nm)

300 320 340 360 380 400

0

20

40

60

80

100

120

140

160

0 - 6.0M GdmCl

Wavelength (nm)

300 320 340 360 380 400

0

20

40

60

80

100

0 - 6.0M GdmCl

A

B

Wavelength (nm)

300 320 340 360 380 400

0

20

40

60

80

100

120

140

160

0 - 6.0M GdmCl

Wavelength (nm)

300 320 340 360 380 400

0

20

40

60

80

100

0 - 6.0M GdmCl

A

B


6/14

Figure 5.5:Tryptophan fluorescence. Panel A: Tryptophan fluorescence intensity of

-crystallin variants at 335nm plotted as function of GdmCl. Fraction of foldedmolecules was calculated by taking the ratio of intensity at 335 nm in absence and

presence of respective GdmCl concentration. Panel B: wavelength maxima ( max) of

-crystallin variants as a function of GdmCl. Data are average of four experiments.

GdmCl [M]

0 1 2 3 4

Fractionfolded

0.5

0.6

0.7

0.8

0.9

1.0

1.1

ABL3:1

1:3

1:1

lamda max

GdmCl [M]

0 1 2 3 4

max

335

340

345

350

355

L3: 11: 31: 1

GdmCl [M]

0 1 2 3 4

Fractionfolded

0.5

0.6

0.7

0.8

0.9

1.0

1.1

ABL3:1

1:3

1:1

GdmCl [M]

0 1 2 3 4

Fractionfolded

0.5

0.6

0.7

0.8

0.9

1.0

1.1

ABL3:1

1:3

1:1

lamda max

GdmCl [M]

0 1 2 3 4

max

335

340

345

350

355

L3: 11: 31: 1

lamda max

GdmCl [M]

0 1 2 3 4

max

335

340

345

350

355

L3: 11: 31: 1


7/14

Figure 5.6: Representative ANS-fluorescence profile ofpanel A: B andpanel B: 3:1

( A: B), as a function of Gdmcl. Concentration of GdmCl is indicated in numbers.

Wavelength (nm)

400 420 440 460 480 500 520 540

0

100

200

300

400

500

1

2

3

4,5,6

1 : 0 M

2 : 0.5M

3 : 1.0M

4 : 1.5M

5 : 2.0M6 : 3.0M

Wavelength (nm)

400 420 440 460 480 500 520 540

0

100

200

300

400

500

2

3

1

4

5

6

1 : 0 M

2 : 0.5M

3 : 1.0M

4 : 1.5M

5 : 2.0M

6 : 3.0M

A

B

Wavelength (nm)

400 420 440 460 480 500 520 540

0

100

200

300

400

500

1

2

3

4,5,6

1 : 0 M

2 : 0.5M

3 : 1.0M

4 : 1.5M

5 : 2.0M6 : 3.0M

Wavelength (nm)

400 420 440 460 480 500 520 540

0

100

200

300

400

500

2

3

1

4

5

6

1 : 0 M

2 : 0.5M

3 : 1.0M

4 : 1.5M

5 : 2.0M

6 : 3.0M

A

B


8/14

Figure 5.7: ANS Fluorescence. ANS fluorescence intensity of -crystallin variants as

a function of GdmCl. Fraction of folded molecules was calculated by taking the ratio ofintensity at 475 nm in absence and presence of respective GdmCl concentration. Data

are average of four experiments.

GdmCl [M]

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Fra

ctionfolded

0.0

0.5

1.0

1.5

2.0

2.5

3.0L3:11:31:1


9/14

Figure 5.8:Secondary structure of -crystallin variants.Far-UV CD spectrum of -crystallin variants as a function of GdmCl. Each spectrum

is an average of five accumulations.

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

B

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A

aL far uv Aug2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

L

3,1 far uv unfold Jul2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M4.0 M

6.0 M

3:1

1,1 far uv Aug2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

1:1


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

1:3

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A

Far UV aA profile Jul2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A

mdeg

mdeg

mdeg

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

B

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A

aL far uv Aug2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

L


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M4.0 M

6.0 M

3:1

1,1 far uv Aug2008

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

1:1


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

1:3

Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A


Wavelength (nm)

210 220 230 240 250

Mdeg

-15

-10

-5

0

0 M

2.0 M

4.0 M

6.0 M

A

mdeg

mdeg

mdeg


10/14

Figure 5.9: Tertiary structure of -crystallin variants.Near-UV CD spectrum of -crystallin variants as a function of GdmCl. Each spectrum is the average of fiveaccumulations

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

A

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

2D Graph 3

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

L

2D Graph 4

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

3:1

2D Graph 5

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

1:3

2D Graph 6

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

1:1

mdeg

mdeg

mdeg

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

A

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

2D Graph 3

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

L

2D Graph 4

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

3:1

2D Graph 5

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

1:3

2D Graph 6

Wavelength (nm)

240 260 280 300 320 340 360

Mdeg

-3

-2

-1

0

1

2

0 M

0.5 M

1.0 M

1.5 M

1:1

mdeg

mdeg

mdeg


11/14

Figure 5.10: Light scattering of -crystallin variants at 85oC

Time (sec)

0 500 1000 1500 2000 2500 3000

Lig

htscattering,

360nm

0.08

0.09

0.10

0.11

0.12

0.13

0.14

L

A3:1

1:1

1:3

B


12/14

Figure 5.11: HPLC profile of goat TSP and TSP-alpha

HPLC profile of TSP and TSP-alpha on TSKG3000SWXL column depicts the depletion of -

crystallin from TSP upon ultracentrifugation.

Volume (ml)0 5 10 15 20 25 30

A280nm

-2

0

2

46

8

10

12

14

16

18

20

Goat TSP

Goat TSP-alpha


13/14

Figure 5.12: Light scattering of goat lens TSP at 85oC

Panel A: Light scattering of goat TSPalpha in the absence (trace 1) and presence of A-

homopolymer(trace 2), heteropolymer with 3:1 A to B ratio (w/w) ratio (trace 3)

and TSP control (trace 4).Panel B: Aggregation pattern of goat TSPalpha in the absence

(trace 4) and presence of 0.05 (trace 3), 0.1 (trace 2) and0.15 mg/ml (trace 1) B-crystallin.

Time (sec)

0 200 400 600 800 1000

LightScattering(36

0nm)

0.0

0.5

1.0

1.5

2.0

1

2

3

4

B

Time (sec)

0 200 400 600 800 1000

Lihtscatterin

,360nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

2

1

3,4

A

Time (sec)

0 200 400 600 800 1000

LightScattering(360nm)

0.0

0.5

1.0

1.5

2.0

Time vs TSP-

Time vs TSP- with aB 50

Time vs TSP- with aB 100Time vs TSP- with aB150

1

2

3

4

Time (sec)

0 200 400 600 800 1000

LightScattering(36

0nm)

0.0

0.5

1.0

1.5

2.0

1

2

3

4

B

Time (sec)

0 200 400 600 800 1000

LightScattering(36

0nm)

0.0

0.5

1.0

1.5

2.0

1

2

3

4

B

Time (sec)

0 200 400 600 800 1000

Lihtscatterin

,360nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

2

1

3,4

A

Time (sec)

0 200 400 600 800 1000


0.0

0.5

1.0

1.5

2.0

Time vs TSP-



1

2

3

4

Time (sec)

0 200 400 600 800 1000

Lihtscatterin

,360nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

2

1

3,4

A

Time (sec)

0 200 400 600 800 1000

Lihtscatterin

,360nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

2

1

3,4

A

Time (sec)

0 200 400 600 800 1000


0.0

0.5

1.0

1.5

2.0

Time vs TSP-



1

2

3

4


14/14

stability chapter iv figures

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