Figure 5.

1: Differential scanning calorimetric (DSC) profiles of -crystallin

variants. 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 got
precipitated.
8 12

A B
10
6

8
Cp (kcal/mole/ C)

Cp (kcal/mole/ C)
o

o
4
6

4
2

0
0

10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90
o o
T e m p e ra tu re ( C ) T e m p e ra tu re ( C )
8 8

L 3 :1
6 6
Cp (kcal/mole/ C)

Cp (kcal/mole/ C)
o

4 4

2 2

0 0

10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90
o
T e m p e ra tu re ( C ) o
T e m p e ra tu re ( C )
12 8

1:3 1:1
10
6
8
Cp (kcal/mole/oC)

Cp (kcal/mole/oC)

6 4

4
2

0
0

10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90
o o
Temperature ( C) Temperature ( C)
Table 5.1: Thermodynamic data from DSC profiles derived using the MicroCal
Origin software. Data are mean±SE (n=3).

αL αA αB 1:1 3:1 1:3

Scan rate at 30/hr
Tm oC 58.95 55.17 62.10 59.72 58.31 62.07
±0.12 ±0.24 ±0.12 ±0.34 ±0.12 ±0.41
ΔH 45.08 62.95 44.20 80.70 72.10 70.31
(kcal.mol-1) ±0.18 ±0.40 ±0.59 ±0.63 ±0.20 ±1.24
Scan rate at 60/hr
Tm oC 58.99 56.12 62.98 61.01 59.84 62.20
±0.22 ±0.21 ±0.12 ±0.12 ±0.17 ±0.12
ΔH 49.48 64.25 61.59 64.58 60.56 94.33
(kcal.mol-1) ±0.23 ±0.18 ±1.30 ±0.30 ±0.27 ±1.98
Scan rate at 90/hr
Tm oC 60.18 56.86 62.90 61.21 59.16 62.51
±0.14 ±0.11 ±0.21 ±0.03 ±0.03 ±0.31
ΔH 45.22 67.37 62.70 73.75 51.78 69.29
(kcal.mol-1) ±0.30 ±0.13 ±0.13 ±0.42 ±0.37 ±1.67
Figure 5.2: Size-exclusion chromatography (SEC) of -crystallin variants. Elution
profiles of normal (continuous line) and preheated (dashed line) -crystallin variants on
a TSKG4000SWXL gel filtration HPLC column. Elution positions of standard
molecular weight markers, thyroglobulin (650 kDa; position-1), ovalbumin (150 kDa;
position-2) and BSA (67 kDa; position-3) are indicated on the top.

1 2 3

A



L
A280nm







normal
preheated

0 5 10 15 20
Time(min)
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 mean±SD (n=3)

Sample Hydrodynamic Radii – Rh (nm)

o
unheated at 25 C 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 ±1.34
3:1 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
Figure 5.4: Representative tryptophan fluorescence profiles of panel A: A and panel
B: 3:1 (A: B), as a function of GdmCl.

160
A
0 - 6.0M GdmCl
Fluorescence intensity

140

120

100

80

60

40

20

0
300 320 340 360 380 400
Wavelength (nm)

100 B
0 - 6.0M GdmCl
Fluorescence intensity

80

60

40

20

0
300 320 340 360 380 400
Wavelength (nm)
Figure 5.5: Tryptophan fluorescence. Panel A: Tryptophan fluorescence intensity of
-crystallin variants at 335nm plotted as function of GdmCl. Fraction of folded
molecules 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.

Frac unfold AVG

A

1.1
A
1.0 B
Fraction folded

L
3:1
0.9 1:3
1:1
0.8

0.7

0.6

0.5

0 1 2 3 4

GdmCl [M]
lamda max
B
355

350
 max

345



340 L


335 

0 1 2 3 4

GdmCl [M]
Figure 5.6: Representative ANS-fluorescence profile of panel A: B and panel B: 3:1
(A:B), as a function of Gdmcl. Concentration of GdmCl is indicated in numbers.

500
A 2
1: 0M
Fluorescence intensity

2: 0.5M
400 3: 1.0M
4: 1.5M
1 5: 2.0M
300 6: 3.0M
3

200

100
4,5,6

400 420 440 460 480 500 520 540

Wavelength (nm)

500 B
1: 0M
2 2: 0.5M
Fluorescence intensity

400 3: 1.0M
4: 1.5M
3
5: 2.0M
300 6: 3.0M
1
200
4
100 5
6

400 420 440 460 480 500 520 540

Wavelength (nm)
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 of
intensity at 475 nm in absence and presence of respective GdmCl concentration. Data
are average of four experiments.

ANS Frac unfold

3.0


2.5
L
Fraction folded


2.0 

1.5

1.0

0.5

0.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

GdmCl [M]
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.
Far UV aA profile Jul2008
aB Far UV Jul2008

0 A B
0

-5 -5
mdeg
Mdeg

Mdeg
-10 -10 Far UV aA profile Jul2008

0M 0 0M A

2.0 M -5 2.0 M

M deg
4.0 M - 10 4.0 M
-15 6.0 M -15 - 15
6.0 M
0M
2.0 M
4.0 M
6.0 M

210 220 230 240 250

Wavelength (nm)

210 220 230 240 250 210 220 230 240 250

Wavelength (nm) Wavelength (nm)

aL far uv Aug2008 3,1 far uv unfold Jul2008

L 3:1
0 0

-5 -5
mdeg
Mdeg

Mdeg

-10 Far UV aA prof ile Jul2008

-10 Far UV aA profile Jul2008

0 0M A
0
A

-5 2.0 M -5
0M
M deg

M deg
2.0 M
-10 4.0 M 0M
- 10

4.0 M 0M
2.0 M 2.0 M

-15 -15 6.0 M 4.0 M

6.0 M
-15 - 15
6.0 M
4.0 M
6.0 M

210 220 230 240 2 50 210 220 230 240 250

Wavelength (nm) Wavelength (nm)

210 220 230 240 250 210 220 230 240 250

Wavelength (nm) Wavelength (nm)

1,3 far uv unfold Jul2008 1,1 far uv Aug2008

1:3 1:1
0 0
mdeg

-5 -5
Mdeg
Mdeg

Far UV aA prof ile Jul2008 Far UV aA p rofile Jul2008

-10 0
A -10 0
A

-5 0M -5
0M
Mdeg
M deg

-10 2.0 M -10

2.0 M
0M 0 M

-15 4.0 M
2.0 M
4.0 M
6.0 M -15 4.0 M 2.0 M
4.0 M
6.0 M

6.0 M -15 6.0 M

-15
210 220 230 240 2 50 210 220 230 240 250
Wavelength (nm) Wavelength (nm)

210 220 230 240 250 210 220 230 240 250
Wavelength (nm) Wavelength (nm)
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 five
accumulations

2D Graph 1
2D Graph 2

2
2
A 
1 1
mdeg

0 0
Mdeg

M deg
-1 0M -1
0.5 M 0M
1.0 M 0.5 M
-2 1.5 M -2 1.0 M
1.5 M

-3 -3
240 260 280 300 320 340 360 240 260 280 300 320 340 360

Wavelength (nm) Wavelength (nm)

2D Graph 3
2D Graph 4

2 2
L 3:1
1 1
mdeg

0 0
M deg

Mdeg

-1 -1
0M 0M
0.5 M 0.5 M
-2 1.0 M -2 1.0 M
1.5 M 1.5 M

-3 -3
240 260 280 300 320 340 360 240 260 280 300 320 340 360
Wavelength (nm) Wavelength (nm)
2D Graph 6
2D Graph 5

2
2
1:1
1:3
1
1
mdeg

0 0
Mdeg
Mdeg

-1 -1

0M 0M
0.5 M 0.5 M
-2 -2 1.0 M
1.0 M
1.5 M 1.5 M

-3 -3
240 260 280 300 320 340 360 240 260 280 300 320 340 360
Wavelength (nm) Wavelength (nm)
Figure 5.10: Light scattering of -crystallin variants at 85oC

0.14
B

0.13
Light scattering, 360 nm

1:3
0.12

0.11

1:1
0.10
A
0.09 3:1
L

0.08
0 500 1000 1500 2000 2500 3000
Time (sec)
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.

20
18 Goat TSP
-alpha
Goat TSP
16
14
12
A 280 nm

10
8
6
4
2
0
-2
0 5 10 15 20 25 30
Volume (ml)
Figure 5.12: Light scattering of goat lens TSP at 85oC
Panel A: Light scattering of goat TSP–alpha 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 TSP–alpha in the absence
(trace 4) and presence of 0.05 (trace 3), 0.1 (trace 2) and 0.15 mg/ml (trace 1) B-
crystallin.

0.30
A
2.0
Light scattering, 360 nm

1
0.25 1
1.5

0.20
1.0
2
0.15
0.5
g6
3m
n
0)

2
h
gS
ce
a
t
tn
r
i(

3
L
it

0.
4
0.10
3,4
0 20 40 60 80 10 0
Time(sec)
0.05
Time vs TSP-
Time vs TSP-with aB 50
Time vs TSP-with aB 10
Time vs TSP-with aB150
0.00
0 200 400 600 800 1000
Time (sec)
L ig h t S c a tte rin g (3 6 0 n m )

2.0 B
1
1.5

1.0
2

0.5
3
4
0.0
0 200 400 600 800 1000
Time (sec)

Time vs TSP-
Time vs TSP- with aB 50
Time vs TSP- with aB 100
Time vs TSP- with aB150