Supplemental Materials
Temperature Shift Effect on the Chlorobaculum tepidum Chlorosomes
Joseph Kuo-Hsiang Tang1,*, Xing Xu2, Guillermo M. Muhlmann1, Farrokh Zare1, Yadana Khin1,
Sun W. Tam1,2
1
School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA, and
2
Nuclea Biotechnologies Inc., Pittsfield, MA 01201, USA
Running title: Temperature shift on chlorosomes
*To whom correspondence should be addressed: Tel: 1-614-316-7886, Fax: 1-508-793-8861, Email: jtang@clarku.edu
1
Fig. S1. Growth curves and absorption spectra of constant temperature and temperature
shift cultures measured at different temperatures. The absorption spectra of constant
temperature cultures at 50 oC (A) and cultures down-shifted to 30 oC (B), and culture growth
curves for constant temperature cultures at 50 oC and cultures down-shifted to 30 oC (C) and
constant temperature cultures at 30 oC and cultures up-shifted to 50 oC (D) are shown.
A
B
1.8
2
1.6
o
1.8
25 oC
o
1.6
30 oC
1.4
40 C
1.2
50 oC
25 C
1.4
40 C
o
50 C
Abs.
Abs.
1.2
1
0.8
1
0.6
0.8
0.4
0.6
0.4
0.2
400
500
600
700
800
o
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
C
D
3
Cultures down-shifted
3.5
Constant temperature
to 30 oC
3
Constant temperature
Cultures up-shifted to
OD
625
625
cultures at 50 oC
2.5
OD
cultures at 30 oC
2.5
2
50 oC
2
1.5
1.5
1
1
0.5
0.5
0
0
0
10
20
30
40
50
60
70
0
10
20
30
40
Time (hr)
Time (hr)
2
50
60
70
Fig. S2. Absorption spectra of chlorosomes with versus without DTT addition. The
absorption spectra of chlorosomes from constant temperature cultures at 50 oC (A) and from
cultures down-shifted from 50 oC to 30 oC (B) with versus without 100 mM DTT are shown.
The chlorosomes samples without versus with DTT are noted as “aerobic” and “reduced”
samples, respectively.
A
B
0.08
0.08
Aerobic
Aerobic
Reduced
0.06
Abs.
0.04
0.04
0.02
0.02
0
400
Reduced
Abs.
0.06
500
600
700
0
400
800
500
600
700
Wavelength (nm)
Wavelength (nm)
3
800
Fig. S3. Absorption (A) and CD spectra (B) of intact chlorosomes, BChl c monomers and
aggregates. BChl c monomers were acquired through methanol extraction of chlorosomes.
A
B
40
2
Intact chlorosomes
BChl c monomer

670 nm

Abs.
BChl c monomers
BChl c aggregates
30
CD (mdegree)
1.5
748 nm
1
20
10
0
-10
0.5
-20
0
400
-30
480
560
640
720
800
400
880
Wavelength (nm)
500
600
700
wavelength (nm)
4
800
Fig. S4. CD spectra of chlorosomes and BChl c aggregates form various cultural conditions.
The CD spectra of chlorosomes and BChl c aggregates from constant temperature cultures at 50
o
C (A) and from cultures down-shifted to 30 oC (B), concentration series measurements for
chlorosomes from constant temperature cultures at 50 oC (C), cultures down-shifted to 30 oC (D),
BChl c aggregates from constant temperature cultures at 50 oC (E) and BChl c aggregates from
cultures down-shifted to 30 oC (F) are shown.
A
B
100
100
Chlorosomes
Chlorosomes
BChl c aggregates
BChl c aggregates
50
CD (mdeg)
CD (mdeg)
50
0
-50
-50
-100
0
400
500
600
700
-100
800
400
Wavelength (nm)
500
600
700
800
Wavelength (nm)
C
D
40
no dilution
dilute 1.5-fold
dilute 3-fold
dilute 6-fold
dilute 12-fold
20
10
40
CD (mdeg)
CD (mdeg)
30
0
-10
-20
no dilution
2-fold dilution
4-fold dilution
8-fold dilution
16-fold dilution
20
0
-20
-30
-40
-40
400
500
600
700
400
800
Wavelength (nm)
500
600
700
800
Wavelength (nm)
E
F
40
20
32
CD (mdeg)
16
CD (mdeg)
no dilution
dilute x 2-fold
dilute x 4-fold
dilute x 8-fold
24
8
0
-8
16
No dilution
12
dilute x 2-fold
dilute x 4-fold
8
4
0
-4
-16
-8
-24
400
500
600
700
-12
800
Wavelength (nm)
400
500
600
700
Wavelength (nm)
5
800
Fig. S5. HPLC chromatograms in LC/MS analyses. The HPLC chromatographs monitoring at
640 nm for the pigments extracted from chlorosomes from constant temperature cultures at 50 oC
(upper panel) and from cultures down-shifted from 50 oC to 30 oC (lower panel). Injection
volume was 5 μL per sample. Elution/acquisition time for peaks 1, 2, 3, 4, 5 and 6 is 4.9, 5.3,
5.7, 6.2, 6.7 and 7.2 (min), respectively.
2
Chlorosomes from constant
temperature cultures at 50 oC
3
1
4
2
Chlorosomes from cultures
down-shifted to 30 oC
5
6
3
1
4
5
Acquisition time (min)
6
6
Fig. S6. MS/MS spectra of bacteriochlorophyll c isolated from the Cba. tepdium
chlorosomes. The data for m/z 793.5 (A), 807.5 (B), 823.5 (C), 839.5 (D) and 853.5 (E) are
shown.
A
793.5
MS/MS data for m/z 793.5
589.2
B
603.3
MS/MS data for m/z 807.5
807.5
7
C
619.3
MS/MS data for m/z 823.5
823.5
D
MS/MS data for m/z 839.5
635.3
839.5
8
E
649.3
MS/MS data for m/z 853.5
853.5
9
Fig. S7. CD spectra of aerobic chlorosomes and buffer at different temperatures.
CD
spectra of buffer (20 mM Tris-HCl at pH 7.8) at 25, 45, 65 and 85 C (A), chlorosomes from
constant temperature cultures at 50 oC incubating at 85 oC versus cooling to 25 oC from 85 oC
(B).
o
A
B
20
15
30
Buffer
85 oC
o
45 C
25 C
o
65 C
o
20
85 C
CD (mdeg)
CD (mdeg)
10
o
5
0
-5
85 oC --> 25 oC
10
0
-10
-10
-15
-20
-20
400
500
600
700
800
400
Wavelength (nm)
500
600
700
Wavelength (nm)
10
800