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

2. 2 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 : 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. 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. 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. 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 Ca 2+ buffer that controls the Ca 2+ concentration profile inside the cell

6. 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. 7 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

8. 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. 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. 10 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.

11. 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. 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. 13 Spectrum in ethylene glycol All the further experiments were conducted in 30% ethylene glycol. β α

14. 14 Stability of Stains all Concentration (M)= OD at 578nm- OD at 700nm/1.13*10 5 Day 0 – 0.256mM Day 1 – 0.28mM Day 2 – 0.323mM Day 3 – 0.323mM Day 4 – 0.315mM

15. 15 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. AB C

16. 16 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. AB C

17. 17 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. AB C

18. 18 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. AB C

19. 19 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. AB C

20. 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. 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. 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. 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. 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. 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. 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. 27 Comparison of interaction of BSA with Stains-all/Exp 2,3 and 4.

28. 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. 29 Acknowledgements  Dr. Judith  Harpreet  David

30. 30 Thank you very much for your attention!