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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)
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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
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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
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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
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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
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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
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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
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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
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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
1,3 far uv unfold Jul2008
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
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
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
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
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
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
1,3 far uv unfold Jul2008
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
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
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
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
mdeg
mdeg
mdeg
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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
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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
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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
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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
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
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
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
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