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Lab Report: GARP 2 & Stains-All studies

Fernanda Balem Department of Pharmacology 10/17/05. Lab Report: GARP 2 & Stains-All studies. What are GARP Proteins?.  GARPs are G lutamic A cid R ich P roteins  They are exclusively expressed in rod photoreceptor cells  There are 3 GARP-Proteins :

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Lab Report: GARP 2 & Stains-All studies

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  1. Fernanda Balem Department of Pharmacology 10/17/05 Lab Report:GARP 2 & Stains-All studies

  2. 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).

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. GARP2 Vectors, Expression and Purification Baculovirus Expression Transient Transfection Stable Transfection Generation of recombinant Baculovirus and Gene Expression with the Bac-to-Bac Expression System by Invitrogen Construction of Plasmid Containing Synthetic Bovine Gene in pMT4 Construction of GARP2 Expression Plasmid using pACMV-tetO Expression in Cos-1Cells Expression in Sf-9 Insect Cells Expression in HEK293S Cells 6; 7 Figure 3: Strategy for Large Scale Expression of GARP 2

  8. 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.

  9. 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).

  10. 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 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).

  11. 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

  12. 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.

  13. Spectrum in ethylene glycol β α All the further experiments were conducted in 30% ethylene glycol .

  14. 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

  15. A B C 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.

  16. A B C 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.

  17. A B C 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.

  18. A B C 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.

  19. A B C 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.

  24. 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.

  25. 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.

  26. 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.

  27. Comparison of interaction of BSA with Stains-all/Exp 2,3 and 4.

  28. 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!!!!!

  29. Acknowledgements • Dr. Judith • Harpreet • David

  30. Thank you very much for your attention!

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