Download Characterization of the chimeric seven

Appl Microbiol Biotechnol (2013) 97:819–828
DOI 10.1007/s00253-012-4452-y
METHODS AND PROTOCOLS
Characterization of the chimeric seven-transmembrane
protein containing conserved region of helix C–F
of microbial rhodopsin from Ganges River
Ah Reum Choi & Se Jun Kim & Byung Hoon Jung &
Kwang-Hwan Jung
Received: 20 March 2012 / Revised: 9 September 2012 / Accepted: 19 September 2012 / Published online: 15 November 2012
# Springer-Verlag Berlin Heidelberg 2012
Abstract Proteorhodopsin (PR) is a light-driven proton
pump that has been found in a variety of marine bacteria.
Recently, many PR-like genes were found in non-marine
environments. The goal of this study is to explore the
function of rhodopsins that exist only as partial proteoopsin genes using chimeras with marine green PR (GPR).
We isolated nine partial genes of PR homologues using
polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which
has earned them the name “CFR, Chimeric Freshwater
Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices
C to F and inserted the isolated conserved partial sequences.
When expressed in E. coli, we could observe light-driven
proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs
ranged between 143 and 333 ms at pH 10; their absorption
maxima were between 515 and 522 nm at pH 7. We can
guess that the function of native rhodopsin, a retinal protein
of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep
reporting partial PCR-based opsin sequences to finally characterize their proteins.
Keywords Opsin . Membrane protein . Proton pumping .
Environmental genomics . Freshwater rhodopsin
A. R. Choi : S. J. Kim : B. H. Jung : K.-H. Jung (*)
Department of Life Science and Institute of Biological Interfaces,
Sogang University,
Shinsu-Dong 1, Mapo-Gu,
Seoul 121-742, South Korea
e-mail: [email protected]
Introduction
Proteorhodopsin (PR) was first discovered 10 years ago by
Beja et al. (Atamna-Ismaeel et al. 2008) in uncultivated
marine γ-proteobacteria of SAR86 group (Beja et al. 2000,
2001; Brown and Jung 2006). It has been shown that PR is a
type I rhodopsin which functions as a light-driven proton
transporter using retinal photo-isomerization and subsequent protein conformational changes. Since then, a large
number of PR homologues were found in bacteria throughout the marine photic zone such as Monterey Bay (Eastern
Pacific Ocean), Hawaii Ocean Time (HOT, Central North
Pacific Ocean), Antarctic Peninsula, Mediterranean Sea,
Red Sea, Sargasso Sea, Arctic Ocean, and Antarctic Sea
ice (Beja et al. 2001; de la Torre et al. 2003; Frigaard et al.
2006; Jung et al. 2008; Koh et al. 2010; Man et al. 2003;
Man-Aharonovich et al. 2004; McCarren and DeLong 2007;
Sabehi et al. 2005, 2003; Venter et al. 2004). Recently, it has
been reported that the genes of the archaeal-type rhodopsins
are present in some non-marine bacteria (Sharma et al.
2008). More divergent proteorhodopsin-related sequences
originating from non-marine organisms like Gloeobacter
violaceus (Nakamura et al. 2003) and Roseiflexus sp. RS-1
(Hanada et al. 2002), bacteria isolated from a calcareous
rock, and a Japanese hot spring have been found as well.
Gloeobacter rhodopsin is characterized as a fast-cycling
rhodopsin capable of light-driven proton transport, similar
to proteorhodopsin (Miranda et al. 2009). Similarly, many
actinorhodopsin genes, proteorhodopsin-like sequences,
were found predominantly in non-marine environments
(Sharma et al. 2009, 2008). The discovery led to novel
understanding of survival strategies and the role of phototrophy in biogeochemical cycles (de la Torre et al. 2003;
Fuhrman et al. 2008). Extensive insight was gained into the
evolutionary relationship between different photosystems
(Frigaard et al. 2006; McCarren and DeLong 2007;
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Sharma et al. 2006; Spudich 2006) and biogeographical
analyses that have shown important differences in PRs in
different geographical and ecological contexts (Papke et al.
2003; Pasic et al. 2005; Sabehi et al. 2003). Many PRs and
their homologues have been discovered by polymerase
chain reaction (PCR)-based gene survey using degenerate
primers, or through genome sequencing of bacterial artificial
chromosome, fosmids, and with environmental shotgun libraries (Beja et al. 2001; de la Torre et al. 2003; Frigaard et
al. 2006; Jung et al. 2008; Koh et al. 2010; Man et al. 2003;
Man-Aharonovich et al. 2004; McCarren and DeLong 2007;
Sabehi et al. 2005, 2003; Venter et al. 2004).
The absorption maxima of PR variants depend on the
places and depth of the ocean where their hosts reside (Beja
et al. 2001; Man et al. 2003). PR variants from the surface or
the deep ocean from the same place (e.g., Hawaiian Pacific
Ocean) have different absorption maxima being spectrally
tuned to usable light in their environment (Beja et al. 2001).
PR from Monterey Bay and HOT_0m (surface) has absorption maxima tuned for green light (525 nm), whereas PRs
from the Antarctic Ocean and Hot_75m4 (75 m deep) have
blue-shifted absorption maxima (490 nm). This is achieved by
a substitution of a single amino acid residue at the position 105
(Leu in green light-absorbing proteorhodopsin (GPR) and Gln
in blue light-absorbing proteorhodopsin (BPR)), which thereby functions as a spectral tuning switch (Beja et al. 2001; Man
et al. 2003; Sabehi et al. 2007). There are major differences
between GPR (MBP) and BPR (Hot_75m4) including the
absorption maxima, photochemical reactions, and proton
pumping efficiency (Chomczynski and Sacchi 1987). The
natural occurrence and vertical distribution of green- versus
blue-light-absorbing PRs in the oceans can therefore at least
partly be accounted for by adaptation to the prevailing light
spectra (Pasic et al. 2005; Wang et al. 2003).
In this work, we have used degenerate PR gene primers
(Rusch et al. 2007) to positively identify PR-bearing operational taxonomic units from the surface of the Ganges
River. We constructed a cassette with GPR sequence missing helices C to F and inserted the found fragments in it. In
addition to chimeric proteins (named CFR, Chimeric
Freshwater Rhodopsin), modified GPR and chimeric GPR/
BPR (as controls) were characterized by biophysical methods such as absorption spectroscopy, light-induced difference spectroscopy, flash-induced photolysis, and lightdriven proton pumping assays.
Materials and methods
Sampling and extraction of total DNA
A water sample (1 L) from the surface of the Ganges River in
the city of Varanasi, India (25°16′55′′N, 82°57′23′′E), was
Appl Microbiol Biotechnol (2013) 97:819–828
collected in December 2008 and processed immediately in
Seoul, Korea. The water sample was filtered first through a
10-μm-pore-size filter and through a 0.2-μm-pore-size filter.
Total genomic DNA was extracted from each filtrated fraction
using the Trizol methods (Chomczynski and Sacchi 1987).
PCR amplification from total genomic DNA from Ganges
River, cloning, and DNA sequencing
For detection of PR homologue genes in environmental
genomic DNA samples from the filtrate, a multiplex PCR
analysis with degenerate primers was performed. Primers
were designed using conserved helix C and helix F regions
of GPR. Primers used in this study are listed in Table 1.
PCRs for PR homologues were performed using Taq DNA
polymerase (Vivagen, Korea) in twice. PCR amplification
was carried out with a total volume of 25 μl containing 1 μl
(∼1 to 5 ng/μl) of template DNA, 200 μΜ deoxynucleoside
triphosphates (dNTPs), 1.5 mM MgCl2, 5 pmol primers, and
0.5 units of Taq DNA polymerase. The amplification comprised the following program: an initial step at 95 °C for
1 min and then 40 cycles at 95 °C for 1 min, 50 °C for
1 min, and 72 °C for 2.5 min. At the end of every PCR, a
post-elongation step at 72 °C for 5 min was carried out. PCR
products were visualized by gel electrophoresis. The product (330 bp) was excised from the gel and purified with the
Qiagen gel extraction kit (Qiagen, Germany). The purified
DNA fragments were cloned with the T-Blunt PCR cloning
kit for DNA sequencing (SolGent, Korea) and sequenced.
Design of chimeric proteins
To express the full protein from the partial PR homologue
sequences, we designed a cassette for chimeric rhodopsin.
Table 1 Proteorhodopsin primers used in this study
Primer
Direction
Sequence (5′–3′)
(A)RYIDW
(C)RYIDW
LRYIDW
LRYVDW
FRYIDW
LRYVDWILT
Forward
Forward
Forward
Forward
Forward
Forward
GWAIYP
WFLLVGWAIYP
Reverse
Reverse
GWVIYP
GWSIYP
Reverse
Reverse
AGNTAYATHGAYTGG
CGNTAYATHGAYTGG
CTCCGTTATATHGAYTGG
CTCCGTTATGTTGATTGG
TTNMGNTAYATHGAYTGG
CTCCGTTATGTTGATTGGA
TTTTAACA
CGGGTAAATCGCCCAACC
CGGGTAAATCGCCCAACC
AACTAGAAGGAACA
NGGRTADATNACCCANCC
NGGRTADATNSWCCANCC
Parenthesis for the first two amino acids means dNTP of start
Reported in Sabehi et al. 2007
Appl Microbiol Biotechnol (2013) 97:819–828
The pKA001 plasmid template contains a partial proteorhodopsin gene with N-terminal and C-terminal regions
(Fig. 1). At the RYIDW region of helix C, we modified
the DNA sequence to insert KpnI restriction enzyme site by
site-directed mutagenesis. As a result, the RYIDW region is
changed to the RYLDW. In the other conserved region,
GWAIYP, the NgoMIV restriction enzyme site was created
by site-directed mutagenesis from (604th bp) GTAGGT to
GCCGGC (Fig. 1). Then, helix C–F regions of PR homologue sequences, BPR, BR, and NpSRII were cloned into
this cassette.
Expression and purification of CFRs
To express chimeric fresh water rhodopsins, we transformed
the plasmid into Escherichia coli UT5600 strain. The
pKA001 plasmid contains a chimeric freshwater rhodopsin
gene. The transformed cells were induced with 1 mM IPTG
(Applichem, USA) and 5 μM all-trans retinal (Sigma, USA)
for 6 h at 35 °C. The collected cells were sonicated (Branson
sonifier 250), and the membrane fraction was treated with
1 % n-dodecyl-β- D -maltopyranoside (DM) (Anatrace,
USA), and the protein fraction was bound to the Ni+2–
NTA resin and eluted with 0.02 % DM and 250 mM imidazole (Sigma, USA).
Absorption spectroscopy and pKa measurements
Absorption spectroscopy was used to measure absorption
spectra and to calculate pKa values of the Schiff base counterion in purified chimeric freshwater rhodopsins. The absorption spectra were recorded with Shimadzu UV-vis
spectrophotometer (UV-2550) at pHs 4, 7, and 10. In order
to calculate the pKas of the primary proton acceptor, the
spectrum at pH 7.0 was used as a reference, and pH was
lowered from 7.0 to 4.0 and raised from 7.0 to 10. The
corrected ratio of protonated and deprotonated forms at
different pH values was determined as previously described
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(Wang et al. 2003) from the intensities of the absorption
band that appears at λmax of each new component as the pH
is changed. The data were fitted to functions containing
titration components (y0A/(1+10pH−pKa)) using Origin Pro
6.1 (Wang et al. 2003), where A represents the maximal
amplitude of relative absorbance changes.
Proton pumping measurement
Spheroplast vesicles as described (Wang et al. 2003) were
isolated by centrifugation at 30,000×g for 1 h at 4 °C
(Beckman XL-90 ultracentrifuge) and washed with 3 ml of
10 mM NaCl, 10 mM MgSO4·7H2O, 100 μM CaCl2 (Wang
et al. 2003). Samples were illuminated at 100 W/m2 intensity through the short-wave cutoff filter (>440 nm, Sigma
Koki SCF-50S-44Y, Japan) in combination with focusing
convex lens and heat-protecting (CuSO4) filter, and the pH
values were monitored by Horiba pH meter F-51.
Light and laser-induced absorption difference spectroscopy
Light-induced static absorbance changes were measured on
Schinco (Korea) spectrometer. Flash-induced transient absorbance changes were measured for 10,000 ms on RSM
1000 (Olis, USA) spectrometer. The actinic flash was from
an Nd-YAG pulse laser (Continuum, Mini-light II, 532 nm,
6 ns, 25 mJ). Thirty signals were averaged for measuring the
rate of formation and decay of the photo-intermediates. To
measure a clear signal purified membranes were incorporated into 7 % poly-acryl amide gels, which were soaked in
50 mM Tris, 150 mM NaCl at pH 10.0 (Jung et al. 2008).
Nucleotide sequence accession numbers
Partial opsin sequences obtained in this study were deposited in GenBank under the following accession numbers:
JX169790-JX169798.
Results
Chimeric freshwater rhodopsin variants from the Ganges
River
Fig. 1 The schematic for the cassette containing N-terminal and Cterminal parts of GPR, with the topological model of the putative
protein product shown at the bottom. Freshwater rhodopsin partial
gene was inserted into the plasmid at the KpnI and NgoMIV restriction
sites
A total of nine distinct chimeric fresh water rhodopsin
sequences were obtained from 96 of clone samples from
the Ganges River by PCR-based gene survey. We amplified
genomic DNA using the ten PR primer combinations recommended previously by Atamna-Ismaeel et al. 2008. In all
primer combinations (Table 1), these sets ((A)RYIDW, (C)
RYIDW, LRYIDW, LRYVDW, FRYIDW, LRYVDWILT as
forward primer and GWAIYP, WFLLVGWAIYP, GWVIYP,
GWSIYP as reverse primer) were used to amplify the
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Appl Microbiol Biotechnol (2013) 97:819–828
Fig. 2 Phylogenetic tree of
partial fresh water genes in
Ganges River based on
neighbor-joining analysis of
translated nucleotide sequences.
The helix C to F fragments
corresponding to freshwater
opsin were used for analysis.
Analysis was conducted by
using the sequence alignments
by Clustal W. Sequences are
labeled with NCBI database
accession numbers
homologues of PR. Pairs of primer (LRYVDW + GWAIYP,
LRYVDWILT + GWAIYP, LRYVDW + WFLLVGWAIYP,
and LRYVDWILT + WFLLVGWAIYP) gave positive
results. Phylogenetic analysis showed that the freshwater
rhodopsin sequences clustered within groups (Fig. 2). As
shown in Fig. 2, each CFR of our clones is the individual
sequence rather than clusters like putative microbial rhodopsin from lake Kinneret, Israel, putative proteorhodopsins
from Atlantic and Pacific oceans, Antarctic Sea ice, and
marine flavobacteria. The amino acid sequence alignment
shows that nine partial opsin sequences differ from each
other at 60 positions out of 112 residues of the cloned
fragment (Fig. 3). Among these, 39 positions were located
Helix C
Helix D
in the helices E and F, and three positions are in the retinal
binding pocket.
Spectral properties of the CFR/GPR and BPR/GPR chimera
In this study, we have expressed the three of CFR/GPR and
BRP/GPR chimera in E. coli, followed by solubilization
with DM and purification through Ni +2–NTA column.
First of all, the purified modified GPR possesses the λmax
identical to that of the wild-type GPR (522 nm), as shown
for the case of GPR whose gene contains the KpnI/NgoMIV
restriction enzyme sites in Fig. 4. This is also the case for
BPR/GPR (Fig. 4), which shows the identical λmax with the
Helix E
Helix F
* *
*
* *
*
*
* ** ** *
CFR5
LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWAIYP
CFR7
LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWALYP
CFR9
LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWAIYP
CFR3
LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFVLVGWAIYP
CFR2
LRYVDWLLTVPLMCVEFYLITKKVGSTQSLLWKLIAASVGMLVTGYVGEAIYPTESVSWVWGAISGLFYFYIVYLVWFGEVAKLAGNAGPDVAAANKTLAWFVLVGWAIYP
CFR4
LRYVDWLLTVPLMCVEFYLITKKSG-GTTGLLCKMILASVVMLVTGYWGEAGLGN--ATIWGTISAIAYFYIVYEVWMGDVKKLATSAGSAVADANSALGWFVLVGWAIYP
CFR8
LRYVDWLLTVPLMCVEFYLITKKAGGTIGLLWKLIIASIFMLVTGYIGEAMHGQDASSWVWGTISSIGYAYIVWLVWAGDVAKLAKSSSPAVAAANRYLGWFVLVGWAIYP
CFR1
LRYVDWLLTVPLMCVEFYLITKKAG-AKTSLLWKLILASVVMLVTGFFGEATDRGN-SVLWGVISGAAYFYIAYLVWFGEVASLSNTAGPSVAKATRILAWFVLVGWAIYP
CFR6
LRYVDWILTVPLMCVEFYLILKVAG-AKQSLMWKMIILSLVMLVTGYAGETIDRPN-AWLWGLISGIAYFVIVYEIWLGEASKIAQAAGGNVLSAHKILCWFLLVGWAIYP
GPR
93 FRYIDWLLTVPLLICEFYLILAAATNVAGSLFKKLLVGSLVMLVFGYMGEAGIMAAWPAFIIGCLA--WVYMIYELWAGEGKSACNTASPAVQSAYNTMMYIIIFGWAIYP 201
BPR
93 FRYIDWLLTVPLQVVEFYLILAACTSVAASLFKKLLAGSLVMLGAGFAGEAGLAPVLPAFIIGMAG--WLYMIYELYMGEGKAAVSTASPAVNSAYNAMMMIIVVGWAIYP 201
Fig. 3 Amino acid sequences comparison of nine partial freshwater
opsin genes from the Ganges River, GPR, and BPR. Differences
between the freshwater opsins are marked with black boxes. Based
on BR, transmembrane helices and residues in contact with the
**
chromophore retinal are marked with black arrows and asterisks,
respectively. Underlined amino acids are primer sequences. This alignment was made using Clustal W program
Appl Microbiol Biotechnol (2013) 97:819–828
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Fig. 4 Absorption spectra of GPR (w/KpnI and NgoMIV), BPR/GPR, CFR1/GPR, CFR2/GPR, and CFR3/GPR chimeras at different pH values.
Purified rhodopsins were in 50 mM Tris–HCl (pH 7), 150 mM NaCl, and 0.02 % DM
Table 2 The absorption maxima, the major pKas of the Schiff base counterion, and the photocycle kinetics of CFRs
Name
GPR w/KpnI, NgoMIV
BPR/GPR
CFR1/GPR
CFR2/GPR
CFR3/GPR
λmax (pH 4)
λmax (pH 7)
λmax (pH 10)
pKa
M decay (ms)
O decay (ms)
541
515
545
536
535
522
491
522
520
515
519
491
513
513
511
7.6
5.8
6.9
6.1
5.7
36
ND
352
130
130
49
70
263
119
123
The M decays and O decays were fitted to bi-exponential functions and only the rate of major component is shown
ND indicates that the photocycle could not be measured due to the limitation of the instrument
824
wild-type BPR (491 nm) at pH 7. The absorption maxima of
all the CFR isolates were between 515 and 522 nm at pH 7
(Table 2), which falls into the green light region. CFR 1
absorbs light maximally at 545 and 513, at pH 4 and 10,
respectively (Fig. 4). The most blue-shifted CFR variant,
CFR3, displayed λmax 0535 nm at pH 4 and 511 nm at
pH 10 (Fig. 4). The colors of three CFRs isolates at neutral
pH are illustrated in Fig. 4. CFRs were analyzed in this
study contained non-polar methionine at position homologous to 105, similar to GPR with leucine at this position but
unlike BPR which has polar Gln (Fig. 3), suggesting that
they are green-absorbing rhodopsins. Accordingly, the absorption spectra suggest that all CFR isolates from the
Ganges River absorb mainly green light. In the case of
BR/GPR and NpSRII/GPR, it could not form a pigment
with retinal.
Counterion titration and proton pumping activities
The artificial purified CFR variants were titrated to determine the pKa of the major spectral transition corresponding
to the deprotonation of the Schiff base counterion, and only
a major pKa is shown in Table 2. The titration of each
rhodopsin did not fit well to a single pKa, presenting major
and second minor components (Fig. 5). Especially in case of
CFR2, it looks like two almost equal components in Fig. 5.
Among them, the major component was selected by more
fraction value into the Table 2. The pKa values of Asp-97 for
CFRs in DM were 5.7∼6.9, in case CFR2, which is about
one unit lower than that of GPR (7.6).
We made right-side-out vesicles of E. coli (Wang et al.
2003) that contained each CFR for measuring proton transport activity. Pumping activity of each CFR was verified by
measuring pH difference between the spheroplast suspension in the presence and absence of illumination through
short-wave cutoff filter (>440 nm), where the difference of
pH could be converted to the number of transported protons.
CFRs have ability to translocate protons in E. coli spheroplast upon illumination (Fig. 5). At similar pigment concentration of GPR (w/KpnI, NgoMIV), three CFR/GPR
chimeras, and BPR/GPR chimera, the pumping activity of
CFRs is similar to one of GPR. As expected, the pumping
activity of BPR/GPR chimera is lower than that of GPR,
similar to the wild-type BPR (Wang et al. 2003).
Photochemical properties of the CFR/GPR and BPR/GPR
chimeras
PR has a unique photocycle that is comprised of a series of
intermediates, such as blue-shifted M and red-shifted Ointermediates. To study the photocycle of CFRs, lightinduced UV-vis spectroscopy and flash photolysis were
used (Fig. 6). First, we tested if the photocycle is modified
Appl Microbiol Biotechnol (2013) 97:819–828
by the introduction of KpnI and NgoMIV restriction enzyme
sites. The photocycle of modified GPR with two restriction
sites is similar to that of the wild-type GPR. Also, it should
be noted that BPR/GPR exhibits the photocycle similar to
the wild-type BPR. The left panel in Fig. 6 shows the lightinduced difference spectrum of CFRs in the gels at pH 10
over the spectral range from 300 to 800 nm continuous
illumination. Maximal depletion by the light of the original
pigment was observed at 510 nm. These values were almost
the same as the absorption maxima of CFRs at pH 10 shown
in Fig. 4 (513 and 511 nm). At 400 nm, an increase of
absorbance was observed, implying the formation of an
intermediate M of CFR. They all have slower M than
GPR. Also, at 600 nm, an increase of absorbance was
observed, implying the formation of an intermediated O of
CFR. Figure 6, right panel, shows laser-induced absorbance
changes of the CFRs at selected wavelength, 400 nm (dash
line), 510 nm (solid line), and 600 nm (dash–dot–dot line),
which monitor the M intermediate, the depletion of CFR,
and the O intermediate, respectively, at the room temperature. The M-decay rate and O-decay rate were estimated by
double exponential fitting. The main decay time constants
(half time) of the M and O intermediates are presented
Table 2. Although GPR exhibits fast M and O decay
(<50 ms), three green absorbing CFRs from the Ganges
River have slower M and O decay than that of BPR.
Discussion
We have constructed a cassette with GPR sequence missing
helices C to F and inserted the found fragments that contain
the helix C to F in it (Fig. 1). The GPR (w/KpnI, NgoMIV)
and the BPR/GPR chimera showed characteristic absorption
spectra (Fig. 4), indicating no structural perturbation of the
retinal binding region by the chimeric protein.
Microbial rhodopsins from the environmental samples of
fresh water habitats have not been studied yet, although PRs
from the oceans are well-known in terms of their sequences,
absorption maxima, and photochemical reactions (Beja et al.
2001; Kelemen et al. 2003; Man et al. 2003; ManAharonvich et al. 2004; Sabehi et al. 2005). All chimeric
Fig. 5 The left panels show pH dependencies of the relative concen-„
tration of acid versus alkaline forms of the GPR (w/KpnI and NgoMIV), BPR/GPR, CFR1/GPR, CFR2/GPR, and CFR3/GPR chimeras.
Difference spectra were constructed using the spectrum at pH 7.0 as the
reference. The pH of purified His-tagged rhodopsins in 50 mM Tris,
150 mM NaCl, and 0.02 % DDM were adjusted with dilute NaOH or
HCl. pH titration curves indicate the relative concentration of acid
(protonated) form of the pigments. The right panels show proton
transporting activities of GPR (w/KpnI and NgoMIV), BPR/GPR chimera, and CFR/GPR chimeras. Illumination (>440 nm) was applied to
spheroplasts for 60 after 60 s in the dark period (black boxes), and this
cycle was repeated three times. Initial pH values were adjusted to 8.0
Appl Microbiol Biotechnol (2013) 97:819–828
825
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Appl Microbiol Biotechnol (2013) 97:819–828
Appl Microbiol Biotechnol (2013) 97:819–828
ƒFig. 6
On the left panel, light-induced difference spectra of GPR (w/
KpnI and NgoMIV), BPR/GPR chimera, and three CFR/GPR chimeras
in gel at pH 10 are shown over a spectral range from 300 to 800 nm
under the continuous illumination. On the right panel, the O formation
and decay, return to the ground state, and the M decay were measured
at 600, 510, and 400 nm, respectively
freshwater rhodopsin variants in the Ganges River absorb in
the green light region and their amino acid sequences are
more similar to that of GPR rather than BPR (Beja et al.
2001). Although the chimera proteins are successfully
expressed, the pKa values of the Schiff base counterions in
CFRs are similar to that of GPR. In case of CFR3, the value
is lower than that GPR, suggesting a small structural difference around the chromophore between CFR3 and GPR. In
the case of BR/GPR chimera and NpSRII/GPR chimera, the
expression of chimeric proteins was not successful due to
the structural perturbation of the retinal binding region (data
not shown). Also, CFRs must be adapted to its environment,
that of freshwater with pH around 7.0, which is lower than
in the ocean water (around 8.0). All chimeric freshwater
rhodopsins were isolated in this study showed photochemical reactions much slower than that of GPR. Each CFR has
several hundred millisecond photocycle like sensory rhodopsins. In general, slow photocycle might be particularly
important to distinguish transport rhodopsin from sensory
rhodopsin. Though they have slower photocycle kinetics,
interestingly, CFRs have proton pumping activity similar to
that of GPR.
Generally, microbial rhodopsins are widespread all over
the world. However, there are some limitations to get full
sequence of those from nature. Our findings suggested that
this cassette platform exhibits the potential applications to
study function of microbial rhodopsins when we can only
have partial genome. Also, we will provide many investigators using seven transmembrane ion pumping rhodopsins,
the molecular basis of the different spectral properties of
partial proteo-opsins that this method will be very easy to
speculate on given partial proteo-opsin genes made of short
and simple sequences.
Acknowledgments This work was supported by the Research Foundation of Korea grants (331-2008-1-C00242 and 2011–0012320) and
the second stage of Brain Korea 21 graduate fellowship program for
AR Choi, SJ Kim, and BH Jung. We thank Leonid Brown to critical
comments on this research.
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