Transcript Lab Report: GARP 2 & Stains-All studies Fernanda Balem Department of Pharmacology

Lab Report:
GARP 2 & Stains-All studies
Fernanda Balem
Department of Pharmacology
10/17/05
1
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).
2
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;
3
ABCR: Rim ABC transporter.
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
4
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
5
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.
6
Figure 3: Strategy for Large Scale Expression of
GARP 2
GARP2
Vectors, Expression
and Purification
Baculovirus
Transient
Transfection
Expression
Stable
Transfection
Generation of recombinant Baculovirus
and Gene Expression with the Bac-toBac Expression System by Invitrogen
Construction of Plasmid
Containing Synthetic
Bovine Gene in pMT4
Construction of GARP2 Expression
Plasmid using pACMV-tetO
Expression in Cos-1
Cells
Expression in Sf-9
Insect Cells
Expression in HEK293S
Cells 6; 7
7
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.
8
Figure 7: GARP-2 expression and
purification by Sf-9 cells on Stains-All gel
M=Marker,S1= Cells in hypo tonic buffer,S2=S1 cell pellet after 1% DM solubilization, P=
Pellet after S2 centrifugation resuspended in PBS, FT=Flow through, WT= Wash through (20
μl/ sample).
9
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
E5 E6
(4hr)
E4 E4 E5
(ON)
(ON)
75kD
50kD
Garp-2
M=Marker, E1= Elution1, E2 =Elution2, E3=Elution3, E3(4hr)=Elution3 kept at 20°C for 4Hrs,
E4= Elution4, E4(ON)=Elution4 kept at 20°C overnight, E5=Elution5, E5(ON)=Elution5 kept at
20°C overnight, E6=Elution 6. (20μl loaded / elution).
10
Stains all

Metachromatic cationic carbocyanine dye “Stains-all” (1ethyl-2-{3-(1-ethyl-naphthol[1,2-d]thiazoline-2-ylidine)-2methylpropenyl}
 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 Stainsall while others proteins are stained red or pink
11
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.
12
Spectrum in ethylene glycol
β
α
All the further experiments were conducted in
30% ethylene glycol .
13
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/1.13*105
14
Interaction of Polyglutamic acid
(PGA) with Stains-all
A
B
C
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.
15
Interaction of Polyglutamic acid
+ CaCl2 with Stains-all
A
B
C
A- visible spectrum of PGA + CaCl2 with Stains all complexes with 2mM MOPS,30% ethylene glycol,pH 7.2. The dyePGA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively. B- Shows the difference spectra from Stains
16
all/PGA + CaCl2. C - Prominent peaks of difference spectra.
Interaction of Calmodulin with
Stains-all
A
B
C
A- visible spectrum of Calmodulin with Stains all complexes with 2mM MOPS,30% ethylene glycol,pH 7.2. The dyeCalmodulin mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively. B- Shows the difference spectra 17
from Stains all/Calmodulin. C- Prominent peaks of difference spectra.
Interaction of Calmodulin +
CaCl2 with Stains-all
A
B
C
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.
18
Interaction of BSA with Stains-all in
~ 1 hour - Experiment 1
A
B
C
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 19
all/BSA. C- Prominent peaks of difference spectra.
Interaction of BSA+CaCl2 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 20
from Stains all/BSA+CaCl2. C- Prominent peaks of difference spectra.
Interaction of BSA with Stainsall 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.
21
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.22
C- Prominent peaks of difference spectra.
Interaction of BSA with Stainsall (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.
23
C- Prominent peaks of difference spectra.
Interaction of BSA with Stainsall in ~1 hour – Experiment 3
A- visible spectrum of BSA with Stains all complexes with 2mM MOPS,30% ethylene glycol, pH 7.2. The dyeBSA 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.
24
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.
25
Interaction of BSA with Stainsall 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.
26
Comparison of interaction of BSA
with Stains-all/Exp 2,3 and 4.
27
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!!!!!
28
Acknowledgements
Dr. Judith
Harpreet
David
29
Thank you very much for
your attention!
30