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Supporting Information
Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein Eita Sasaki and Donald Hilvert* Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland
Figure S1-5 Table S1-5
S1
(A)
I = 0.21 M
1 hr
AaLS-13 monomer concentration
6 days
1 day (0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
50 M
350 M
1.4 mM
(B)
I = 0.91 M
AaLS-13 monomer concentration
1 hr
6 days
1 day (2)
(0)
(46)
(2)
(2)
(36)
(13)
(24)
(85)
50 M
380 M
2.0 mM
S2
(C)
(85)
Figure S1. Negative-stain TEM analysis of AaLS-13 capsid assembly from pentamers. Typical images of the AaLS-13 samples (A) in 20 mM TrisHCl buffer (pH 7.8) containing 0.2 M NaCl (I = 0.21) and (B) in 20 mM TrisHCl buffer (pH 7.8) containing 0.9 M NaCl (I = 0.91) (magnification: 22,000, scale bar: 200 nm). AaLS-13 monomer concentration (50 M – 2.0 mM) and incubation time (1 h, 1 day, or 6 days) for each image are indicated at left and top of the images, respectively. The protein concentration of each sample was adjusted to 0.04 mg/mL (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Counts of complete capsid particles in larger view (magnification: 8,900, see C for example) are shown at upper right corner of each image. (C) Example of the larger view image for the sample showing 85 capsid particles in squares (2.0 mM AaLS-13, I = 0.91 M, 6 days, magnification: 8,900, scale bar: 500 nm).
S3
1 day
7 days
Ionic strength
I = 0.21 M
I = 0.41 M
I = 0.61 M
I = 0.81 M
Figure S2. Negative-stain TEM analysis of AaLS-13 capsid assembly from pentamers in the presence of GFP(+36) at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.04 mg/mL of AaLS-13 (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S4
1 day
5 days
Ionic strength
I = 0.21 M
I = 0.41 M
I = 0.61 M
I = 0.81 M
Figure S3. Negative-stain TEM analysis of AaLS-13 capsid in the presence of GFP(+36) at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.04 mg/mL of AaLS-13 (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S5
2 days
11 days
Ionic strength
I = 0.21 M
I = 0.40 M
I = 0.59 M
I = 0.78 M
Figure S4. Negative-stain TEM analysis of AaLS-13 capsid in the presence of GFP(+36) and AaLS-13 pentamers at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.08 mg/mL of AaLS-13 (corresponding to 4 uM of the total AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S6
(A)
(B)
(C)
(D)
*
Figure S5. Negative-stain TEM analysis of AaLS-neg capsids (A), AaLS-neg capsids incubated with GFP(+36) (B), or AaLS-neg capsids incubated with AaLS-13 pentamers and GFP(+36) for 1 day (C) or 9 days (D). Typical images of the samples prepared in 20 mM TrisHCl buffer (pH 7.8) with 0.5 M NaCl (I = 051) are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.02 mg/mL of AaLS-neg (corresponding to 1 uM of the AaLS-neg monomer) before analysis. Complete and incomplete double-shells around AaLS-neg capsids are indicated by normal and diamond arrows in yellow, respectively. Assembled AaLS-13 capsid and double-shell around AaLS-13 capsids are indicated by stealth and normal arrows in white, respectively. Triple-shell capsid is marked with *.
S7
Table S1. Occurrence of multishell assembly from AaLS-13 pentamer and GFP(+36) 1 day incubation
7 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
not applicable (0)
not applicable (0)
I = 0.41 M
65% (40)
80% (35)
I = 0.61 M
39% (176)
58% (135)
I = 0.81 M
7.2% (69)
53% (186)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-4 TEM images (magnification 8,900) for each sample.
Table S2. Occurrence of multishell assembly from AaLS-13 capsid and GFP(+36) 1 day incubation
5 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
6.5% (77)
3.4% (29)
I = 0.41 M
9.0% (111)
3.2% (31)
I = 0.61 M
9.6% (521)
16% (140)
I = 0.81 M
2.3% (481)
5.7% (335)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-6 TEM images (magnification 8,900) for each sample.
S8
Table S3. Occurrence of multishell assembly from AaLS-13 capsid, pentamer, and GFP(+36) 2 day incubation
11 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
2.7% (438)
1.4% (633)
I = 0.40 M
34% (787)
33% (454)
I = 0.59 M
24% (547)
22% (614)
I = 0.78 M
6.7% (629)
7.0% (894)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-4 TEM images (magnification 8,900) for each sample.
Table S4. Occurrence of multishell assembly from AaLS-neg capsid, AaLS-13 pentamer, and GFP(+36) 1 day incubation
9 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
AaLS-neg core
17% (211)
21% (195)
AaLS-13 core
3.8% (26)
13% (64)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from three TEM images (magnification 22,000) for each sample.
Table S5. Triangulation number (T) and estimated capsid diameter (d) based on 16-nm capsid (T = 1) and the quasi-equivalence theory1 T
1
3
4
7
9
12
d (nm)
16
27
32
42
48
55
1
13 58 2
According to the Caspar and Klug’s quasi-equivalence theory, the icosaherally symmetric capsid with triangulation number T (T = h + hk + k2, h > 0, k ≥ 0, h and k are zero or a positive integer: note that there are no allowable T numbers between 4 and 7 or between 9 and 12) is composed of 60T proteins (reference 28). Thus, the capsid surface area should be proportional to T, and the diameter (d) should be proportional to square root of T (reference 14). The diameters for T = 3, 4, 7, 9, 12, and 13 states were calculated based on the size of AaLS-wt (d = 16 nm, T = 1).
S9
Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein Eita Sasaki and Donald Hilvert* Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland
Figure S1-5 Table S1-5
S1
(A)
I = 0.21 M
1 hr
AaLS-13 monomer concentration
6 days
1 day (0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
50 M
350 M
1.4 mM
(B)
I = 0.91 M
AaLS-13 monomer concentration
1 hr
6 days
1 day (2)
(0)
(46)
(2)
(2)
(36)
(13)
(24)
(85)
50 M
380 M
2.0 mM
S2
(C)
(85)
Figure S1. Negative-stain TEM analysis of AaLS-13 capsid assembly from pentamers. Typical images of the AaLS-13 samples (A) in 20 mM TrisHCl buffer (pH 7.8) containing 0.2 M NaCl (I = 0.21) and (B) in 20 mM TrisHCl buffer (pH 7.8) containing 0.9 M NaCl (I = 0.91) (magnification: 22,000, scale bar: 200 nm). AaLS-13 monomer concentration (50 M – 2.0 mM) and incubation time (1 h, 1 day, or 6 days) for each image are indicated at left and top of the images, respectively. The protein concentration of each sample was adjusted to 0.04 mg/mL (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Counts of complete capsid particles in larger view (magnification: 8,900, see C for example) are shown at upper right corner of each image. (C) Example of the larger view image for the sample showing 85 capsid particles in squares (2.0 mM AaLS-13, I = 0.91 M, 6 days, magnification: 8,900, scale bar: 500 nm).
S3
1 day
7 days
Ionic strength
I = 0.21 M
I = 0.41 M
I = 0.61 M
I = 0.81 M
Figure S2. Negative-stain TEM analysis of AaLS-13 capsid assembly from pentamers in the presence of GFP(+36) at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.04 mg/mL of AaLS-13 (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S4
1 day
5 days
Ionic strength
I = 0.21 M
I = 0.41 M
I = 0.61 M
I = 0.81 M
Figure S3. Negative-stain TEM analysis of AaLS-13 capsid in the presence of GFP(+36) at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.04 mg/mL of AaLS-13 (corresponding to 2 uM of the AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S5
2 days
11 days
Ionic strength
I = 0.21 M
I = 0.40 M
I = 0.59 M
I = 0.78 M
Figure S4. Negative-stain TEM analysis of AaLS-13 capsid in the presence of GFP(+36) and AaLS-13 pentamers at various ionic strength. Typical images of the samples are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.08 mg/mL of AaLS-13 (corresponding to 4 uM of the total AaLS-13 monomer) before analysis. Complete and incomplete double-shell capsids are indicated by normal and diamond arrows, respectively.
S6
(A)
(B)
(C)
(D)
*
Figure S5. Negative-stain TEM analysis of AaLS-neg capsids (A), AaLS-neg capsids incubated with GFP(+36) (B), or AaLS-neg capsids incubated with AaLS-13 pentamers and GFP(+36) for 1 day (C) or 9 days (D). Typical images of the samples prepared in 20 mM TrisHCl buffer (pH 7.8) with 0.5 M NaCl (I = 051) are shown (magnification: 22,000, scale bar: 200 nm). The protein concentration of each sample was adjusted to 0.02 mg/mL of AaLS-neg (corresponding to 1 uM of the AaLS-neg monomer) before analysis. Complete and incomplete double-shells around AaLS-neg capsids are indicated by normal and diamond arrows in yellow, respectively. Assembled AaLS-13 capsid and double-shell around AaLS-13 capsids are indicated by stealth and normal arrows in white, respectively. Triple-shell capsid is marked with *.
S7
Table S1. Occurrence of multishell assembly from AaLS-13 pentamer and GFP(+36) 1 day incubation
7 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
not applicable (0)
not applicable (0)
I = 0.41 M
65% (40)
80% (35)
I = 0.61 M
39% (176)
58% (135)
I = 0.81 M
7.2% (69)
53% (186)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-4 TEM images (magnification 8,900) for each sample.
Table S2. Occurrence of multishell assembly from AaLS-13 capsid and GFP(+36) 1 day incubation
5 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
6.5% (77)
3.4% (29)
I = 0.41 M
9.0% (111)
3.2% (31)
I = 0.61 M
9.6% (521)
16% (140)
I = 0.81 M
2.3% (481)
5.7% (335)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-6 TEM images (magnification 8,900) for each sample.
S8
Table S3. Occurrence of multishell assembly from AaLS-13 capsid, pentamer, and GFP(+36) 2 day incubation
11 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
I = 0.21 M
2.7% (438)
1.4% (633)
I = 0.40 M
34% (787)
33% (454)
I = 0.59 M
24% (547)
22% (614)
I = 0.78 M
6.7% (629)
7.0% (894)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from 2-4 TEM images (magnification 8,900) for each sample.
Table S4. Occurrence of multishell assembly from AaLS-neg capsid, AaLS-13 pentamer, and GFP(+36) 1 day incubation
9 day incubation
% multishell1 (total particle counts2)
% multishell1 (total particle counts2)
AaLS-neg core
17% (211)
21% (195)
AaLS-13 core
3.8% (26)
13% (64)
1
[% multishell] = [counts of multishell particles] / [total particle counts] 100
2
Particles were counted from three TEM images (magnification 22,000) for each sample.
Table S5. Triangulation number (T) and estimated capsid diameter (d) based on 16-nm capsid (T = 1) and the quasi-equivalence theory1 T
1
3
4
7
9
12
d (nm)
16
27
32
42
48
55
1
13 58 2
According to the Caspar and Klug’s quasi-equivalence theory, the icosaherally symmetric capsid with triangulation number T (T = h + hk + k2, h > 0, k ≥ 0, h and k are zero or a positive integer: note that there are no allowable T numbers between 4 and 7 or between 9 and 12) is composed of 60T proteins (reference 28). Thus, the capsid surface area should be proportional to T, and the diameter (d) should be proportional to square root of T (reference 14). The diameters for T = 3, 4, 7, 9, 12, and 13 states were calculated based on the size of AaLS-wt (d = 16 nm, T = 1).
S9
