Structural Determinats Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria Page: 3 of 27
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oligonucleotides (all of the oligonucleotides
used in this work are described in Supp Table
1). To obtain double OCP mutants
overexpressing the mutated OCP, the mutated
slr1963 gene was cloned in an overexpressing
plasmid in which the slr1963 gene is under the
control of the psbAII promoter. The
construction of the plasmid was described in
(19). The plasmids containing the mutated
slr1963 genes under the control of the psbAII
promoter were used to transform single OCP
mutants of Synechocystis by double
recombination (Supp. Fig 1).
To confirm the introduction of the
different mutations in the genomic
Synechocystis DNA and complete segregation
of mutants, PCR analysis and digestion by
specific restriction enzymes were performed.
Supp. Fig. 2A shows that amplification of the
genomic region containing the sir1963 and
slr1964 genes (in the psbAII locus (lanes 1-4)
or in the slr1963 locus (lanes 5-9) using the
synthetic psbal and psba2 oligonucleotides or
car6 and car7 oligonucleotides gave fragments
of 1.5 kb or 2.2 kb respectively in the wildtype
and of 3.5 kb and 4.3 respectively in the
mutants containing the antibiotic cassettes. No
traces of the wildtype fragment were detected
in the mutants indicating complete segregation.
The presence of the different mutations was
detected by digestion with restrictions enzymes
(Supp. Figs. 2B and 2C). The presence of the
Y44S and Wi 10F mutation was confirmed by
the appearance of the supplementary NheI
restriction site and the disappearance of the
KpnI restriction site respectively. Digestion of
the amplified DNA fragments by BsmI
indicated the presence of the R155L mutation.
The presence of the mutations was confirmed
by DNA sequencing.
The construction of the His-tagged
OCP, the overexpressing His-tagged OCP,
His-tagged W 110S OCP and the
overexpressing His-tagged W110S OCP were
described in (19).
Purification of the Orange Carotenoid
Protein-- The purification of Synechocystis
OCP was performed as previously described
(19). Briefly, mutant cells (1 mg Chl ml-1) in
Tris-HCl pH=8 buffer were broken in dim light
using a French Press. The membranes were
pelleted and the supernatant was loaded on a
column of Ni-Probond resin (Invitrogen). The
OCP was further purified on a Whatman DE-
52 cellulose column. Additional details of the
purification are described in (19). The protein
was stored in the dark at 4 C.
Absorbance and fluorescence measurements--
Cell absorbance was monitored with an
UVIKONXL spectrophotometer (SECOMAN,
Als). Chlorophyll content was determined in
methanol using the extinction coefficient at
665 nm of 79.24 mg mL- cm-. The orange to
red photoconversion was monitored in a
Specord S600 (Analyticjena, France)
spectrophotometer during illumination of the
OCP with 1200 mol photons m 2 s- of blue-
green light (400-550 nm) at 12 C. Cell
fluorescence was monitored with a pulse
amplitude modulated fluorometer
(101/102/103-PAM; Walz, Effelrich,
Germany). All measurements were carried out
in a stirred cuvette of 1 cm diameter at growth
temperature (32 C). Cells were pre-adapted to
low irradiance of blue-green light (400-550
nm, 80 mol photons m 2 s-) for about 1 min;
then, fluorescence quenching was induced by
740 mol photons m2 s of blue-green light
for about 200 s. Saturating pulses (2000 mol
photons m2 s 1) were applied to measure the
maximal fluorescence levels, Fm', in light-
adapted samples,. Application of such pulses
that transiently close all the Photosystem II
centers serves to distinguish between
photochemical quenching and non-
OCP Immunodetection--Total cell protein was
analyzed by SDS-PAGE on a 12%
polyacrylamide/2 M urea (Supp. Fig. 3) in a
TRIS/MES system (40). The OCP protein was
detected by a polyclonal antibody against OCP
were extracted from OCP proteins as
previously described (41). Liquid
chromatography-mass spectrometry analysis
was performed as described in (42).
Sequence Analysis-- The OCP logo was
produced using an alignment of 28 OCP
orthologs (reciprocal best BLAST hits to
Synechocystis OCP) generated in Tcoffee
coffee/index.html ). The hmmer package of
tools on the Mobyle server at
http://mobyle.pasteur.fr were used to generate
an HMM from the alignment. The HMM was
built using hmmbuild and then calibrated using
hmmcalibrate. The LogoMat-M server at
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Wilson, Adjele; Kinney, James N.; Zwart, Petrus H.; Punginelli, Claire; D'Haene, Sandrine; Perreau, Francois et al. Structural Determinats Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria, article, April 1, 2010; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1012404/m1/3/: accessed September 21, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.