Lab Report: GARP 2

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Lab ReportGARP 2 Stains-All studies

• Fernanda Balem
• Department of Pharmacology
• 10/17/05

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What are GARP Proteins?

• ? GARPs are Glutamic Acid Rich Proteins
• ? They are exclusively expressed in rod
  photoreceptor cells
• ? There are 3 GARP-Proteins
• i- GARP is a part of the B1a-subunit of the rod
  cGMP-gated channel
• ii- two soluble forms GARP1 and GARP2
• ? They contain no sequence similarity to other
  proteins.
• ? GARP2 is the most abundant GARP-species.
• ? GARP2 is a major protein in rod outer segments
  (ROS).

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Figure 1 Schematic Drawing of the
phototransduction signal cascade

• The plasmamembrane of ROD contains CNG-channels,
  which are kept open in the dark by cGMP. The
  Guanylate Cyclase (GC) synthesizes cGMP from GTP.
  Light activated Rhodopsin (Rh) activates the
  G-protein Transducin (T). Active Transducin
  activates the Phosphodiesterase (PDE). PDE
  hydrolyzes cGMP to GMP. The decrease of the cGMP
  concentration leads to the closure of the
  CNG-channels. The cation influx decreases, and
  the membrane hyperpolarisates. GARP proteins are
  localized at the rim region of the ROD disc
  membranes. B1 and A1 CNGB1a and CNGA1 subunit of
  the CNG-channel PDE phosphodiesterase GC
  guanylate cyclase ABCR Rim ABC transporter.

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Figure 2 Schematic Drawing of GARP-Proteins
R1-R4 repeats CaM Calmodulin binding domain
1-6 TM domains with the pore region between TM5
and TM6 cGMP cGMP binding domain Glu glutamic
acid rich region numbers below the schemes aa
which are different at the C-terminus numbers
over aa numbers
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Proposed function of GARP-proteins
? They may use the repeat-region to organize an
adaptional signalling complex to regulate the
high cGMP turnover during daylight . ? They may
cap Peripherin-2 complexes at the rim region 5. ?
They may tether the CNG-channel to the rim region
enforcing a ring like distribution of the
channel. ? GARP2 molecules could serve as
entropic bristles that control the entry of other
proteins into the space between disc and plasma
membrane. ? The high density of negatively
charged glutamate residues may serve as a
low-affinity Ca2 buffer that controls the Ca2
concentration profile inside the cell
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Aim

• ? The structural analysis by NMR may improve the
  understanding of the function of GARP-proteins.
• ? To investigate if Stains all dye could be used
  to explore the conformations of GARP-protein.

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Figure 3 Strategy for Large Scale Expression of
GARP 2
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Figure 4 Transient Expression of StrepTag-GARP2
in COS-1 Cells
The highest amount of recombinant protein was
achieved using 6 h of DNA followed by 2 h of
chloroquine incubation . Cells gave maximum yield
at 72 hours after transfection.
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Figure 7 GARP-2 expression and purification by
Sf-9 cells on Stains-All gel
MMarker,S1 Cells in hypo tonic buffer,S2S1
cell pellet after 1 DM solubilization, P Pellet
after S2 centrifugation resuspended in PBS,
FTFlow through, WT Wash through (20 µl/ sample).
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Figure 8 Sf-9 GARP-2 purification stability on
Stains-All gel
Multiple bands on Stains all gel may be different
conformation of Garp-protein.
M E1 E2 E3 E3(4hr) E4 E4(ON) E5
E5(ON) E6
75kD
Garp-2
50kD
MMarker, E1 Elution1, E2 Elution2,
E3Elution3, E3(4hr)Elution3 kept at 20C for
4Hrs, E4 Elution4, E4(ON)Elution4 kept at 20C
overnight, E5Elution5, E5(ON)Elution5 kept at
20C overnight, E6Elution 6. (20µl loaded /
elution).
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Stains all
? Metachromatic cationic carbocyanine dye
Stains-all (1-ethyl-2-3-(1-ethyl-naphthol1,2-d
thiazoline-2-ylidine)-2-methylpropenyl ? It
can bind to highly acidic proteins . ? It can
also be used to distinguish calcium-binding
proteins (CaBP) from others. CaBP are stained
blue or purple by Stains-all while others
proteins are stained red or pink
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Mass spectroscopy
? We performed mass spectroscopy with the samples
from multiple colored bands from the stains all
gel to check if these bands are Garp-2. ? It was
found after computational analysis that some of
the bands were Garp-2. ? It could be
hypothesized that these bands show multiple
conformations of the Garp-2 protein. In order to
investigate further about these conformation, we
are conducting Stains all spectroscopy studies.
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Spectrum in ethylene glycol
ß
a
All the further experiments were conducted in 30
ethylene glycol .
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Stability of Stains all
Day 0 0.256mM Day 1 0.28mM Day 2
0.323mM Day 3 0.323mM Day 4 0.315mM
Concentration (M) OD at 578nm- OD at
700nm/1.13105
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Interaction of Polyglutamic acid (PGA) with
Stains-all
A- visible spectrum of PGA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-PGA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/PGA.
C- Prominent peaks of difference spectra.
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Interaction of Polyglutamic acid CaCl2 with
Stains-all
A- visible spectrum of PGA CaCl2 with Stains
all complexes with 2mM MOPS,30 ethylene
glycol,pH 7.2. The dye-PGA mole ratios are
Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1
respectively. B- Shows the difference spectra
from Stains all/PGA CaCl2. C - Prominent peaks
of difference spectra.
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Interaction of Calmodulin with Stains-all
A- visible spectrum of Calmodulin with Stains all
complexes with 2mM MOPS,30 ethylene glycol,pH
7.2. The dye-Calmodulin mole ratios are Control,
50, 25, 12.5, 6.25, 3.12, 1.56 and 1
respectively. B- Shows the difference spectra
from Stains all/Calmodulin. C- Prominent peaks of
difference spectra.
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Interaction of Calmodulin CaCl2 with Stains-all
A- visible spectrum of Calmodulin CaCl2 with
Stains all complexes with 2mM MOPS,30 ethylene
glycol, pH 7.2. The dye-CAlmodulin mole ratios
are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1
respectively. B- Shows the difference spectra
from Stains all/Calmodulin CaCl2. C- Prominent
peaks of difference spectra.
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Interaction of BSA with Stains-all in 1 hour -
Experiment 1
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSACaCl2 with Stains-all
A- visible spectrum of BSA CaCl2 with Stains
all complexes with 2mM MOPS,30 ethylene glycol,
pH 7.2. The dye-BSA mole ratios are Control, 50,
25, 12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains
all/BSACaCl2. C- Prominent peaks of difference
spectra.
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Interaction of BSA with Stains-all in 1 hour-
Experiment 2
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSA with Stains all (after 24
hours)
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSA with Stains-all (after 48
hours)
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSA with Stains-all in 1 hour
Experiment 3
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSA with Stains-all Experiment 3
(after 24 hours)
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Interaction of BSA with Stains-all in 1 hour
Experiment 4
A- visible spectrum of BSA with Stains all
complexes with 2mM MOPS,30 ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25,
12.5, 6.25, 3.12, 1.56 and 1 respectively. B-
Shows the difference spectra from Stains all/BSA.
C- Prominent peaks of difference spectra.
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Comparison of interaction of BSA with
Stains-all/Exp 2,3 and 4.
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Future plans

• We plan to investigate about the interaction of
  Garp-2 with stains all dye to help us understand
  if this dye could be used as a system to find
  different conformation of the Garp-2 protein.
• We are trying to find optimum buffer conditions
  to concentrate Garp-2 for NMR studies.
• To move to new building how about buying a
  coffee machine!!!!!

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Acknowledgements

• Dr. Judith
• Harpreet
• David

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Thank you very much for your attention!