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Lab Report:GARP 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

GARP2

Vectors, Expression and Purification

Transient Transfection

Stable Transfection

Baculovirus

Expression

Construction of Plasmid Containing Synthetic Bovine Gene in pMT4

Construction of GARP2 Expression Plasmid using pACMV-tetO

Expression in Cos-1 Cells

Expression in HEK293S Cells 6; 7

Generation of recombinant Baculovirus and Gene Expression with the Bac-to-Bac Expression System by Invitrogen

Expression in Sf-9 Insect Cells

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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

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).

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Figure 8: Sf-9 GARP-2 purification & stability on Stains-All gel

75kD

50kD Garp-2

M E1 E2 E3 E3(4hr) E4 E4(ON) E5 E5(ON) E6

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).

Multiple bands on Stains all gel may be different conformation of Garp-protein.

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Stains all

Metachromatic cationic carbocyanine dye “Stains-all” (1-ethyl-2-{3-(1-ethyl-naphthol[1,2-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

All the further experiments were conducted in 30% ethylene glycol .

β

α

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Stability of Stains all

Concentration (M)= OD at 578nm- OD at 700nm/1.13*105

Day 0 – 0.256mM

Day 1 – 0.28mM

Day 2 – 0.323mM

Day 3 – 0.323mM

Day 4 – 0.315mM

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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.

A B

C

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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.

A B

C

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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.

A B

C

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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.

A B

C

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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.

A B

C

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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 from Stains all/BSA+CaCl2. 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. JudithHarpreetDavid

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Thank you very much for your attention!