Download Transient transfection (Oprian, Molday et al. 1987) was carried with

Glutamic acid rich proteins (Garps): Calcium buffer in visual
phototransduction.
Dhiman, H.K., Balem, F., Plevich, N., Heidemann, K.A. , Benjamin, K., Schwalbe,
H., Klein-Seetharaman, J.
Abstract:
Rod photoreceptors contain an unusual set of three glutamic acid-rich proteins (GARPs):
soluble GARP1 and GARP2, and GARP’, the N-terminal cytoplasmic domain of the B1
subunit of the cGMP-gated ion channel. There are many proposed functions for GARPs,
but the high number of negative charges, in particular, suggests that they serve as calcium
buffer. Previous Calcium titration experiments have already proven that Garp-2 binding
with calcium is high capacity and low affinity (Sabina). To understand the role of Garp-2
protein as a calcium buffer, we expressed Strep-taggged recombinant Garp-2 protein in
three different expression systems: Cos-1, HEK-293-S and sf-9 cells. Recombinant Garp2 was expressed in Cos-1 cells by transient tranfection, HEK-293-S cells by tetracycline
inducible system, and in sf-9 insect cells using Bac-to-Bac Baculovirus expression
system. The Cos-1 and HEK-293S cells yield was low as compared to the insect cells,
which produced 12mg/L Garp-2 protein. The expressed recombinant Garp-2 protein from
insect cells was used to evaluate its calcium binding function using Stains-All dye. The
dye Stains –All binds with acidic proteins and allows visualization of these proteins due
to differential staining. The highly acidic proteins stain blue and less acidic proteins stain
pink. Furthermore, absorption spectral studies were performed to understand the
interaction of Garp-2 protein with Stains –All. Calmodulin and Poly-Glutamic acid were
taken as controls to access the spectra of specific and non-specific calcium binding
proteins. The calmodulin binds with calcium with low capacity and high affinity as
compared to poly-glutamic acid, which has high capacity, low affinity binding sites for
calcium. Garp-2 absorption spectral studies elucidated the fact that Garp-2 has
intermediate behavior of calmodulin and poly Glutamic acid.
1
Introduction:
Visual phototransduction takes place in the outer segments of the rod and cone
photoreceptors. In the plasmamembrane of the outer segment, light reduces the
concentration of cGMP, which in darkness keeps the cationic channel open. Ca2+ plays an
important role in phototransduction by modulating the cGMP-gated channels as well as
cGMP synthesis and breakdown. It also modulates light transduction process by acting at
several sites through Ca2+ -binding proteins (Polans, Baehr et al. 1996). Ca2+ inhibits the
guanylate cyclase (Gorczyca, Gray-Keller et al. 1994; Palczewski, Subbaraya et al. 1994)
and this inhibition of cGMP synthesis is responsible for the observation that calcium
could mimic the effects of light. Ca2+ also reduces the apparent affinity of the cGMPgated channels for cGMP, probably through mediation of calmodulin (Hsu and Molday
1993). Futhermore, Ca2+ inhibits the deactivation of photoactivated rhodopsin through
recoverin, (Erickson, Lagnado et al. 1998) Ca2+ -binding protein. This suggests that
calcium plays an important role in the visual transduction pathway. Here, we propose the
role of GARP as calcium buffer. Specific to rod photoreceptors is an unusual set of three
glutamic acid-rich proteins (GARPs): two soluble forms GARP1 and GARP2 (Sugimoto,
Yatsunami et al. 1991; Colville and Molday 1996; Korschen, Beyermann et al. 1999),
and a third form, which represents the cytoplasmic N-terminal domain (GARP’ part,
almost identical in sequence to GARP1) of the B1 subunit of the cGMP-gated ion
channel (Korschen, Illing et al. 1995). GARPs are characterized by their extremely high
content of glutamate residues (ca. 150 residues in GARP1 and in the GARP’ part)
(Korschen, Illing et al. 1995; Colville and Molday 1996; Korschen, Beyermann et al.
1999).
There are many proposed functions for GARPs, but the high number of negative charges
in particular, suggests that they may serve as a calcium buffer. GARPs organize an
oligomeric protein complex near the cGMP-gated channel (Korschen, Beyermann et al.
1999) and interact with peripherin (Poetsch, Molday et al. 2001), a protein located at the
disc rim (Molday, Hicks et al. 1987). The tethering of the GARP’ part to peripherin is
expected to produce a circular arrangement of cGMP-gated channels in juxtaposition to
the disc rim (Molday, Hicks et al. 1987; Poetsch, Molday et al. 2001; Kaupp and Seifert
2
2002). GARPs, in their native state, are intrinsically unfolded (Batra-Safferling, Abarca
Heidemann et al. 2005). Highly acidic proteins like GARPs bind to metachromatic
cationic carbocyanine dye, Stains–All dye, (1-ethyl-2-{3-(1-ethyl-naphthol[1,2-d]thiazoline2-ylidine)-2-methylpropeny, and allow visualization of these proteins due to differential
staining. It is a convenient way to distinguish between calcium binding proteins (CaBP) and the
others. CaBP are stained blue or purple by Stains-all while others proteins are stained red
or pink (Sharma, Rao et al. 1989). Stains-All is used to detect highly negatively charged
proteins on polyacrylamide gels (Goldberg and Warner 1997). Stains-All dye binds
several Ca2+ -binding proteins, including Calmodulin, troponinC, and Calsequestrin, to
yield spectrally distinct complexes (Campbell, MacLennan et al. 1983). Absorption
spectral studies indicate properties of the complexes formed by interaction of calmodulin
with Stains-All. This complex shows dependence upon Ca2+, and reflect the unusual
structural characteristics of calmodulin, which is the development of the intense J-band in
600-650nm region (Caday and Steiner 1985). To understand the role of GARP-2 proteins
as a calcium buffer, we expressed Strep-tagged recombinant Garp-2 protein in Cos-1
cells by transient tranfection, HEK-293-S cells by tetracycline inducible system, and sf-9
insect cells using Bac-to-Bac Baculovirus expression system. Recombinant Garp-2 is
already expressed in E.coli, but due to low expression of protein, the system could not be
used to study the conformation of the Garp-2 protein (Batra-Safferling, Abarca
Heidemann et al. 2005). The expressed recombinant Garp-2 protein from insect cells
resulted in the highest expression, and was used to evaluate its calcium binding function
using Stains-All dye. Furthermore, absorption spectral studies were performed to
understand the interaction of Garp-2 protein with Stains–All. Calmodulin and PolyGlutamic acid were taken as controls to access the spectra of specific and non-specific
calcium binding proteins. Calmodulin binds with calcium with low capacity and high
affinity. On the other hand, poly-glutamic acid binds calcium with high capacity and low
affinity (Sabina). Experimental studies of interaction of Garp-2 with Stains-All indicate
that the behavior of Garp-2 protein is intermediate to calmodulin, which is specific
calcium binding protein, and polyglutamic acid, a non-specific calcium binding protein.
3
EXPERIMENTAL PROCEDURE
Cloning and expression
GARP-2 gene was expressed in three different systems (Figure1). It was cloned in PMT4 vector for the transient transfection (Oprian, Molday et al. 1987), pACMV-tet-O for the
tetracycline inducible system (Reeves, Kim et al. 2002), and p-Fast Bac-1 for the Bac-toBac baculovirus expression system from Invitrogen (catalog number 10359-016). A total
of 1.25* 109 cells were used to express Garp-2 protein in all the three selected systems.
Transient transfection
Transient transfection (Oprian, Molday et al. 1987) was carried with the cos-1 cells. The
time-course experiment was done to find out the highest protein expression level. To
detect the protein on the western blot, an antibody against full length GARP2 was used
(Korschen, Beyermann et al. 1999).
Tetracycline inducible HEK293S stable cell line
The tetracycline inducible HEK293S stable cell lines for the expression of GARP-2 were
prepared that expressed GARP-2 only in the response to the addition of tetracycline and
sodium butyrate (Reeves, Kim et al. 2002). Different concentrations of the geneticin
(G418) were tested to get distinct colonies producing high yield of GARP-2 protein. The
cell line with the highest yield was perpetuated in 1mg/ml of geneticin and induced after
confluency was achieved. Induction of protein expression was done using tetracycline
(2g/ml) and sodium Butyrate (5mM) followed by harvesting of cells after 2 days.
Bac-Bac baculovirus expression system
Transfection of S. frugiperda cell line Sf9
The Sf-9 cells were seeded (9*105 cells/well) into a tissue culture plate (6-well, 35 mm)
in 2 ml of growth medium containing 50 units/ml penicillin and 50 μg/ml streptomycin
and left for 2 hours at +27°C in a humidified incubator for attachment. After attachment
4
the medium was removed and the cells were washed with 2 ml of un-supplemented
Grace's Medium. The washing medium was removed and 1 ml of DNA:lipid complexes
was added. The cells were incubated for 5 hours at +27°C in a humidified incubator.
Thereafter, the DNA:lipid complexes were carefully removed and 2 ml of complete
growth medium were added to the cells. The first signs of viral infection could be seen
after about 72 hours of incubation. Then the medium containing virus was collected and
stored at +4°C protected from light. Titer of the virus was calculated (BD BacPAK
Baculovirus rapid titer kit, Cat No: K1599-1) followed by making high titer stocks of the
virus. A dose response was established for the virus to determine the optimal infection
parameters for Garp-2 protein expression. We used Multiplicity of infection (MOI) 8,
which resulted in synchronous infection and cells were harvested after 72 hours for
highest yield.
StepTag-GARP2 Protein Purification and Characterization
The purification of the Garp-2 protein was done by affinity chromatography using Streptag affinity column
procured from IBA GmbH, Germany. The cell after transfection
were harvested and re-suspended in 10 mL of hypotonic buffer (100mM Tris-Cl pH 8,
1mM EDTA pH 8) containing PMSF (0.7mM) and Benzamidine (0.005%) and incubated
on ice for 15 minutes. Later, cells were homogenized to get soluble GARP-2 protein.
The slurry of the cells was centrifuged at 35,000 RPM for 45 minutes in 70Ti Beckman
rotor. Soluble Garp-2 was detected in supernatant. The supernatant was removed and
150mM solution of NaCl was added. Soluble Garp-2 supernatant was loaded to the strepgravity flow affinity column, already equilibrated with the washing buffer (100mM TrisCl pH 8, 150 mM NaCl, 1mM EDTA pH 8). The sample was allowed to pass through the
column by gravity followed by twenty column volume washes with the washing buffer
(100mM Tris-Cl pH 8, 150 mM NaCl , 1mM EDTA pH 8). After washing, six elutions
were collected using 2.5mM desthiobiotin in washing buffer. Western blot was done to
verify the purification.
5
Stains-all gel
The GARP-2 protein produced by all the three systems was run on the Stains-All gel
(Figure2). The protein was extracted and purified from equivalent amount 1.25* 109 cells
from all the three systems. After running the SDS Page, the gel were stained with Stainsall dye (Goldberg and Warner 1997).
Spectrometry of protein-Dye complexes: Interaction of Stains-All with calcium
binding protein.
Spectroscopic studies of interaction of Stains –All with calcium binding proteins were
performed. The comparison of purified Garp-2 with Calmodulin(CaM) and Polyglutamic
acid (PGA) was done to identify the behavior of Garp-2 as a calcium binding protein. The
protocol used for preparing the dye solutions and the protein-dye complexes was same as
performed by Caday and Steiner (Caday and Steiner 1985). The Stains - All solutions
were made fresh by dissolving dye in ethylene glycol. The stock solutions of the StainsAll were centrifuged at 50,000rpm in 70Ti rotor in ultracentrifuge to get rid of small
sediment particles. The actual concentration was then determined spectrophotometrically
in ethylene glycol (ε1Mcm,578nm = 1.13 * 105) based on the extinction coefficient of the dye
in ethanol (ε1Mcm,578nm = 1.12 * 105 from Aldrich) (Caday and Steiner 1985). In order to
make the complexes, the calcium binding proteins were dissolved in 2 mM MOPS buffer,
pH 7.2, containing 30% ethylene glycol, to which aliquot of the stock solution of the dye
was added. 20M Stains-All concentration was used to study the Protein-Dye complexes.
The mixture was incubated for 30 minutes in the dark. Different concentration of Dye:
Protein complexes ranging from 50 to 1 were made to find out optimal ratio for the
induction of the interaction between dye and protein. Absorption spectra were recorded
using UV/ VIS Lambda 25 spectrometer from Perkin Elmer.
6
Results & Discussion:
The GARP-2 protein was clearly stained blue with Stains-all (Figure 2); this
suggests that this protein may be a calcium binding protein (CaBP). Most of the CaBPs
have acidic motifs that binds Ca2+, such as calmodulin and calsequestrin (Campbell,
MacLennan et al. 1983). CaBPs stain blue with Stains-all, whereas other proteins stain
pink and the color fades away quickly in the light. Sharma and Balasubramanian (1991)
reported that CaBPs could be separated into three groups from absorption spectra with
Stains-all: proteins that induce a J band (600–650 nm), proteins that induce a γ band
(500–520 nm), and proteins that induce the J and γ bands. It has been proposed that the Jband occurs when the dye is bound to anionic sites that are present in the globular or
compact conformations of the proteins (Sharma Y 1991).Binding of Stains-all with
GARP-2 resulted in the J band at 650 nm (Figure 3). Densitometric scans of Stains-allstained gels revealed that interaction of the dye with Ca2+-binding proteins changed the
absorption spectrum of the dye. The spectral behavior of Garp-2 was seen to be
intermediate between Calmodulin (Figure 4), which is specific calcium binding protein,
and polyglutamic acid, a non-specific calcium binding protein (Figure 5).
All the CaBPs have affinities for Ca2+, which are fine tuned to handle the intracellular
Ca2+ signal, i.e. an oscillating the transient increase of free Ca2+. Calmodulin (CaM) is a
highly conserved EF-hand protein of 148 residues found in all eukaryotic cells and is
responsible for the regulation of well over 100 different target proteins (Yamniuk and
Vogel 2004). Interactions between CaM and the target peptide are mediated almost
exclusively by side chain–side chain interactions. The large hydrophobic clefts exposed
by calcium binding are formed primarily from methionine and other hydrophobic
residues. These hydrophobic residues are surrounded by negatively charged residues,
forming ideal binding surfaces for the interaction with hydrophobic/positively charged
amphipathic target peptides, which form ideal α-helices in the complexes. The induction
of α-helical structure by Ca2+-CaM is believed to be a important step in the activation
process of target proteins (Yuan, Walsh et al. 1999). We performed spectrometry of
Calmodulin-Dye complexes to correlate its behavior to Garp-2-Dye complexes, which we
7
hypothesized as a calcium-binding buffer. The addition of calmodulin to a solution of dye
(20M) in 30% ethylene glycol, 2mM Mops, pH 7.1, results in drastic change in the
absorption spectrum (Caday and Steiner 1985). Calmodulin, specific calcium binding
protein, results in a J-band (600-650nm), at a Dye: Calmodulin ratio of 12.5. At 12.5
Dye:Protein ratio, J-band approaches its maximum intensity and the addition of 1 M
CaCl2 produces significant changes in the spectrum. We see remarkable decrease in the Jband upon addition of calcium (Figure 4).. Similar spectral studies were performed with
the purified recombinant Garp-2 from sf-9 insect cells. The Garp-2 spectra show similar
characteristics of calmodulin (Figure 3), but upon addition of 1 M CaCl2, the decrease
in the J-band was less prominent. This suggests that Garp-2 binds with calcium, but it
may have some shared and different characteristics than calmodulin.
In contrast, Calsequestrin is a high-capacity, intermediate-affinity, calcium- binding
protein (Ikemoto, Nagy et al. 1974; Damiani, Salvatori et al. 1986) present in the lumen
of sarcoplasmic reticulum (SR) of skeletal, cardiac, and smooth muscle. Calsequestrin
has a dual functional role, acting both as a luminal calcium buffer that reduces the
amount of free Ca 2+ inside the SR(Ikemoto, Nagy et al. 1974), and as a modulator of SR
calcium release(Ikemoto, Antoniu et al. 1991). All calsequestrin isoforms are highly
acidic proteins (Yano and Zarain-Herzberg 1994) that at neutral pH have a large calciumbinding capacity (40-55 mol/mol) and which have an intermediate affinity for calcium in
the range of 1 mM in 0.1 M KCI (Ikemoto, Nagy et al. 1974). The nature of calsequestrin
calcium-binding sites remains unknown. Primary sequence determinations show that
calsequestrin contains most of its negatively charged residues localized near the Cterminal region (Fliegel, Ohnishi et al. 1987), suggesting that this region is involved in
calcium binding. It has been proposed that the net charge density rather than distinct
calcium binding sites determine the large calcium-binding capacity of calsequestrin
(Ohnishi and Reithmeier 1987). Calsequestrin undergoes extensive conformational
changes upon cation binding (Ikemoto, Nagy et al. 1974; Ostwald, MacLennan et al.
1974) ; only some of these changes are specific for Ca
2+
(Krause, Milos et al. 1991).
After cation binding, calsequestrin increases its α-helical content and internalizes
aromatic residues and hydrophobic domains. Similarly, Polyglutamic acid is seen to bind
with calcium with low affinity and high capacity ( Sabina). To understand the spectra of
8
the Garp-2 and Stains-All complexes, we conducted similar experiments with PolyGlutamic acid. Interestingly, Garp-2 shows spectral behavior, which is intermediate to
calmodulin and polyglutamic acid. In polyglutamic acid –Stains-All complexes, we
recorded a distinct -band at 535nm. In 30% ethylene glycol in water, the free dye itself
absorbs at 535nm and displays the -band. This indicates that the interaction of
polyglutamic acid with Stains-All occurs upon addition of calcium. Garp-2 like
calsequestrin may undergoes extensive conformational changes upon cation binding. We
see a shift in the -band at 535nm in polyglutamic acid-Stains-All dye complexes upon
addition of 1 M CaCl2. This shift was also recorded in the Garp-2 spectra, in addition to
decrease in the J band at 650nm (Figure 3). This indicates that Garp-2 may have
characteristic intermediate to calmodulin and polyglutamic acid.
Having some shared characteristic calcium binding property as calmodulin, Garp-2 may
act as calcium buffer in visual transduction cascade. The CNG channel of the rod
photoreceptor cell plays a key role in phototransduction by controlling the flow of Na+
and Ca2+ into the rod outer segment (ROS) in response to light-induced changes in
intracellular cGMP concentration. In dark, CNG channels are in open state by reversible
binding of cGMP (Burns 2001), and allows Na+ and Ca2+ ions to enter the outer segment.
This maintains the depolarized state of rod photoreceptors and there is continuous release
of glutamate transmitter at the rod synapse. The Ca2+ concentration in the ROS is kept at
400-500nM by the balanced efflux of Ca2+ from the ROS by the Na/Ca-K exchanger.
Photobleaching of rhodopsin in the disk membrane leads to the activation of the Gprotein mediated visual cascade that results in decrease in the cGMP concentration and
the closure of the CNG channel. The closure of the CNG channel stops the influx of Na+
and Ca2+ ions (Stryer 1986; Pugh and Lamb 1990) and the corresponding
hyperpolarization of the rod cells inhibits the release of glutamate at the synapse. The
closure of the CNG channel also decreases the intracellular Ca2+ concentration to below
100nM as the Na/Ca-K exchanger continues to extrude Ca2+ from the ROS (Yau and
Nakatani 1985; Schnetkamp, Basu et al. 1989). The rod photoreceptor cell returns to its
dark state following photoexcitation through a series of biochemical reactions (Pugh,
Nikonov et al. 1999; Burns 2001).
9
Inactivation of the Rhodopsin is achieved by rhodopsinkinase catalized phosphorylation
followed by binding of arrestin. This results in the return of the phophodiesterase (PDE)
to prebleach activity. Finally, the cGMP concentration is restored to its dark state level
through Ca2+ -dependent activation of guanylate cyclase and CNG channel again opens
(Koch and Stryer 1988). The decrease in the intracellular Ca2+ following illumination
initiates multiple negative feedback mechanisms that contribute to photorecovery (Pugh,
Nikonov et al. 1999; Burns 2001). These mechanism involve Ca2+ binding proteins that
respond to the changes in free intracellular Ca2+ levels (Polans, Baehr et al. 1996;
Palczewski, Polans et al. 2000). Guanylate cyclase modulation by Guanylate cyclase
activating protein (GCAPS) is a major Ca2+ dependent mechanism (Mendez, Burns et al.
2001). Finally, Calmodulin binding to the channel alters the sensitivity of the channel to
cGMP (Hsu and Molday 1993). There could be other unidentified calcium binding
proteins, which may effect in the control of rod channel sensitivity. The Garp-2 is seen to
have some shared spectral properties with Calmodulin (Fig3). Therefore, Garp-2 may
have buffering action in the visual transduction cascade. The cGMP-gated channel is
highly permeable to Ca2+ ions and contributes to 15% of the dark current through cGMPchannel (Nakatani and Yau 1988). The high density of negatively charged glutamate
residues in Garp-2 protein may serve as a low affinity Ca2+ buffer that controls the Ca2+
concentration inside the cell.
10
References
Batra-Safferling, R., K. Abarca Heidemann, et al. (2005). "Glutamic acid-rich proteins of
rod photoreceptors are natively unfolded." J Biol Chem.
Burns, M. (2001). "Activation, deactivation, and adaptation in vertebrate photoreceptor
cells. ." Annu Rev Neurosci 24: 779-805.
Caday, C. G. and R. F. Steiner (1985). "The interaction of calmodulin with the
carbocyanine dye (Stains-all)." J Biol Chem 260(10): 5985-90.
Campbell, K. P., D. H. MacLennan, et al. (1983). "Staining of the Ca2+-binding proteins,
calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine
dye "Stains-all"." J Biol Chem 258(18): 11267-73.
Colville, C. A. and R. S. Molday (1996). "Primary structure and expression of the human
beta-subunit and related proteins of the rod photoreceptor cGMP-gated channel."
J Biol Chem 271(51): 32968-74.
Damiani, E., S. Salvatori, et al. (1986). "Characteristics of skeletal muscle calsequestrin:
comparison of mammalian, amphibian and avian muscles." J Muscle Res Cell
Motil 7(5): 435-45.
Erickson, M. A., L. Lagnado, et al. (1998). "The effect of recombinant recoverin on the
photoresponse of truncated rod photoreceptors." Proc Natl Acad Sci U S A
95(11): 6474-9.
Fliegel, L., M. Ohnishi, et al. (1987). "Amino acid sequence of rabbit fast-twitch skeletal
muscle calsequestrin deduced from cDNA and peptide sequencing." Proc Natl
Acad Sci U S A 84(5): 1167-71.
Goldberg, H. A. and K. J. Warner (1997). "The staining of acidic proteins on
polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in
combination with silver nitrate." Anal Biochem 251(2): 227-33.
Gorczyca, W. A., M. P. Gray-Keller, et al. (1994). "Purification and physiological
evaluation of a guanylate cyclase activating protein from retinal rods." Proc Natl
Acad Sci U S A 91(9): 4014-8.
Hsu, Y. T. and R. S. Molday (1993). "Modulation of the cGMP-gated channel of rod
photoreceptor cells by calmodulin." Nature 361(6407): 76-9.
Ikemoto, N., B. Antoniu, et al. (1991). "Intravesicular calcium transient during calcium
release from sarcoplasmic reticulum." Biochemistry 30(21): 5230-7.
Ikemoto, N., B. Nagy, et al. (1974). "Studies on a metal-binding protein of the
sarcoplasmic reticulum." J Biol Chem 249(8): 2357-65.
Kaupp, U. B. and R. Seifert (2002). "Cyclic nucleotide-gated ion channels." Physiol Rev
82(3): 769-824.
Koch, K. W. and L. Stryer (1988). "Highly cooperative feedback control of retinal rod
guanylate cyclase by calcium ions." Nature 334(6177): 64-6.
Korschen, H. G., M. Beyermann, et al. (1999). "Interaction of glutamic-acid-rich proteins
with the cGMP signalling pathway in rod photoreceptors." Nature 400(6746):
761-766.
Korschen, H. G., M. Beyermann, et al. (1999). "Interaction of glutamic-acid-rich proteins
with the cGMP signalling pathway in rod photoreceptors." Nature 400(6746):
761-6.
11
Korschen, H. G., M. Illing, et al. (1995). "A 240 kDa protein represents the complete beta
subunit of the cyclic nucleotide-gated channel from rod photoreceptor." Neuron
15(3): 627-36.
Krause, K. H., M. Milos, et al. (1991). "Thermodynamics of cation binding to rabbit
skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding
sites." J Biol Chem 266(15): 9453-9.
Mendez, A., M. E. Burns, et al. (2001). "Role of guanylate cyclase-activating proteins
(GCAPs) in setting the flash sensitivity of rod photoreceptors." Proc Natl Acad
Sci U S A 98(17): 9948-53.
Molday, R. S., D. Hicks, et al. (1987). "Peripherin. A rim-specific membrane protein of
rod outer segment discs." Invest Ophthalmol Vis Sci 28(1): 50-61.
Nakatani, K. and K. W. Yau (1988). "Calcium and magnesium fluxes across the plasma
membrane of the toad rod outer segment." J Physiol 395: 695-729.
Ohnishi, M. and R. A. Reithmeier (1987). "Fragmentation of rabbit skeletal muscle
calsequestrin: spectral and ion binding properties of the carboxyl-terminal
region." Biochemistry 26(23): 7458-65.
Oprian, D. D., R. S. Molday, et al. (1987). "Expression of a synthetic bovine rhodopsin
gene in monkey kidney cells." Proc Natl Acad Sci U S A 84(24): 8874-8.
Ostwald, T. J., D. H. MacLennan, et al. (1974). "Effects of cation binding on the
conformation of calsequestrin and the high affinity calcium-binding protein of
sarcoplasmic reticulum." J Biol Chem 249(18): 5867-71.
Palczewski, K., A. S. Polans, et al. (2000). "Ca(2+)-binding proteins in the retina:
structure, function, and the etiology of human visual diseases." Bioessays 22(4):
337-50.
Palczewski, K., I. Subbaraya, et al. (1994). "Molecular cloning and characterization of
retinal photoreceptor guanylyl cyclase-activating protein." Neuron 13(2): 395404.
Poetsch, A., L. L. Molday, et al. (2001). "The cGMP-gated channel and related glutamic
acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor
disc membranes." J Biol Chem 276(51): 48009-16.
Polans, A., W. Baehr, et al. (1996). "Turned on by Ca2+! The physiology and pathology
of Ca(2+)-binding proteins in the retina." Trends Neurosci 19(12): 547-54.
Pugh, E. N., Jr. and T. D. Lamb (1990). "Cyclic GMP and calcium: the internal
messengers of excitation and adaptation in vertebrate photoreceptors." Vision Res
30(12): 1923-48.
Pugh, E. N., Jr., S. Nikonov, et al. (1999). "Molecular mechanisms of vertebrate
photoreceptor light adaptation." Curr Opin Neurobiol 9(4): 410-8.
Reeves, P. J., J. M. Kim, et al. (2002). "Structure and function in rhodopsin: a
tetracycline-inducible system in stable mammalian cell lines for high-level
expression of opsin mutants." Proc Natl Acad Sci U S A 99(21): 13413-8.
Schnetkamp, P. P., D. K. Basu, et al. (1989). "Na+-Ca2+ exchange in bovine rod outer
segments requires and transports K+." Am J Physiol 257(1 Pt 1): C153-7.
Sharma Y, B. D. (1991). "Stains all is a dye that probes the conformational features of the
calcium binding proteins. In CW Heizmann, ed, Novel Calcium-Binding Proteins.
Springer-Verlag, Berlin." pp51-61.
12
Sharma, Y., C. M. Rao, et al. (1989). "Binding site conformation dictates the color of the
dye stains-all. A study of the binding of this dye to the eye lens proteins
crystallins." J Biol Chem 264(35): 20923-7.
Stryer, L. (1986). "Cyclic GMP cascade of vision." Annu Rev Neurosci 9: 87-119.
Sugimoto, Y., K. Yatsunami, et al. (1991). "The amino acid sequence of a glutamic acidrich protein from bovine retina as deduced from the cDNA sequence." Proc Natl
Acad Sci U S A 88(8): 3116-9.
Yamniuk, A. P. and H. J. Vogel (2004). "Calmodulin's flexibility allows for promiscuity
in its interactions with target proteins and peptides." Mol Biotechnol 27(1): 33-57.
Yano, K. and A. Zarain-Herzberg (1994). "Sarcoplasmic reticulum calsequestrins:
structural and functional properties." Mol Cell Biochem 135(1): 61-70.
Yau, K. W. and K. Nakatani (1985). "Light-induced reduction of cytoplasmic free
calcium in retinal rod outer segment." Nature 313(6003): 579-82.
Yuan, T., M. P. Walsh, et al. (1999). "Calcium-dependent and -independent interactions
of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with
calmodulin." Biochemistry 38(5): 1446-55.
13
Figure 1: Western blot analysis of
StrepTag-Garp-2 expression from
Bac-Bac
baculovirus expression system from sf-9 cells, trasient transfection from cos-1 cells and
stable HEK293S cells. To detect the protein an antibody against full length GARP2 was
used.
14
Figure 2: The Stains-All gel of purified recombinant Garp-2 protein HEK293S, Cos-1
and sf-9 cells.
15
Figure 3: The visible spectrum of recombinat Garp-2 from sf-9 cells and Stains-All
complexes at Dye:Protein ratio of 12.5 in 2mM Mops, 30% ethylene glycol, pH
7.2, 23oC.
Garp2
0.5
Garp2/CaCl2
0.4
0.3
0.2
0.1
Wavelength
16
694
680
666
652
638
624
610
596
582
568
554
540
526
512
498
484
470
456
442
428
414
0
400
Absorbance
.
0.6
Figure 4: The visible spectrum of calmodulin-Stains-All complexes at Dye:Protein ratio
of 12.5 in 2mM Mops, 30% ethylene glycol, pH 7.2, 23oC.
1.2
calmo
calmo/CaCl2
0.8
0.6
0.4
0.2
Wavelength
17
694
680
666
652
638
624
610
596
582
568
554
540
526
512
498
484
470
456
442
428
414
0
400
absorbance
.
1
Figure 5: The visible spectrum of polyglutamic acid-Stains-All complexes at Dye:Protein
.
ratio of 12.5 in 2mM Mops, 30% ethylene glycol, pH 7.2, 23oC.
1.2
PGA
PGA/CaCl2
0.8
0.6
0.4
r
0.2
673
642
612
581
550
520
489
459
428
0
400
absorbance
1
Wavelength
18
19
20