SUPPORTING INFORMATION
Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2microglobulin as a simple tool for misfolding and stability studies
Laura Bertoletti 1, Federica Bisceglia 1, Raffaella Colombo 1, Sofia Giorgetti 2, Sara Raimondi 2, P. Patrizia Mangione 2,3
and Ersilia De Lorenzi 1
1
Department of Drug Sciences, University of Pavia, Pavia, Italy.
2
Department of Molecular Medicine, University of Pavia, Pavia, Italy.
3
Centre for Amyloidosis and Acute Phase Proteins, University College London, London, UK
CE-UV analysis of D76Nβ2-m at increasing concentrations
1
A
mAU
120
1
1
100
80
1
2
42 µM
2
60
1
35 µM
2
28 µM
40
1
2
20
24 µM
19 µM
2
15 µM
2
0
6
B
600
10
12
30
Area counts
400
min
Peak 2
Peak 1
y = 12.179x - 12.239
R² = 0.9963
500
Area counts
8
300
200
100
25
20
15
10
y = 0.7403x - 3.1415
R² = 0.9265
5
0
0
0
10
20
30
40
50
0
10
Concentration (µM)
C
30
40
50
600
Peak 1+2
y = 12.868x - 14.54
R² = 0.9951
500
Area counts
20
Concentration (µM)
400
300
200
100
0
0
10
20
30
Concentration (µM)
40
50
Fig S1. (A) Electropherograms of D76Nβ2-m at increasing concentrations. (B) linearity of normalized areas for
each electrophoretic peak, including magnification for data related to peak 2; (C) linearity of the normalized
area sum. These data support that protein is fully soluble over this concentration range and that the
intermediate state is not over populated under these conditions. Experimental conditions as reported in
Materials and Methods.
CE-UV analysis of wt and D76Nβ2-m under partially denatured conditions: evaluation of different
chaotropic agents.
ACN
mAU
mAU
1
Urea
1
mAU
60
60
wt
Urea
HCl
2
50
700
50
1
1
Urea
600
1
500
40
40
ACN
0
20
1
33.3 mM
50%
10
X
2
0
6
mAU
6
D76N
80
70
60
60
2
50
40
Urea
20
10
2
0%
0
6
min
1
300
2
1
2
200
6.5
2
20
0
1
400
30
X
10
500
1
40
30
50%
8
min
mAU
50
1
ACN
6
1
90
70
0M
8
min
1
80
100 3 M
0
mAU
90
200 6 M
2
0 mM
8
min
X
20 mM
8
300
X
0%
7 min 7.5
1
20
10
400
2
30
1
2
2
2
30
2
8
33.3 mM
0 mM
6
7
100
2
20 mM
min
3M
2
0 0M
min
8
6 min
Figure S2. These data demonstrate that denaturants other than ACN induce a reduction of the total area
and/or of peak efficiency, especially at higher concentrations of denaturant
Table S1: Themodynamic stability under different experimental conditions as reported in the literature for
wt β2-m and variants
Reference
[13]
[30]
Isoform
Cm a)
[GdnHCl]
ΔG° (H2O) b)
(kcal mol -1)
Protein
Concentration
µM
wt
2.3
6.3 (±0.2)
16.8
H31Y
2.9
7.4 (±0.1)
16.8
ΔN3
1.9
4.4 (±0.2)
16.8
ΔN6
1.65
3.8 (±0.09)
16.8
wt
H31S
H31Y
ΔN3
ΔN6
R3A
2.3
2.5
2.9
1.9
1.65
2.2
6.3 (±0.2)
6.9 (±0.3)
7.8 (±0.4)
4.4 (±0.2)
3.8 (±0.09)
5.9 (±0.2)
2.11
2.11
2.11
2.2
2.24
2.12
wt
1.8 (±0.1)
5.3 (±0.5)
1.68
[29]
[23]
W60G
2.5 (±0.1)
6.6 (±0.8)
1.7
wt
2.0 (±0.2)
5.7 (±0.4)
1.68
D76N
1.15 (±0.1)
3.0 (±0.15)
1.68
Solubilizing
solvent
T
°C
Method
20 mM
sodium
phosphate
buffer
pH=7.4
20
far-UV
circular
dichroism
20
fluorescence
30
fluorescence
30
fluorescence
20 mM
sodium
phosphate
buffer
pH=7.3
10 mM
sodium
phosphate
buffer
pH=7.4
10 mM
sodium
phosphate
buffer pH=7.4
a) Denaturant concentration at the midpoint of the unfolding transition.
b) Free energy of unfolding in the absence of denaturant.