Reference no: EM132927551
Structural Requirements of the Photoreceptor Phosphodiesterase γ -Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin
Xiu-Jun Zhang, Nikolai P. Skiba, and Rick H. Cote
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, pp. 4455-4463, February 12, 2010
1. What does the abbreviation PDE6 stand for?
2. The authors do not explicitly describe the diesterase reaction carried out by PDE6. Write down the reaction of PDE6 with cAMP.
3. The first paragraph of the introduction describes the vertebrate visual signalling pathway that is activated by the absorption of light by 11-cis-retinal. Draw a labelled, schematic diagram that illustrates the events in this pathway
4. In your own words, describe the structure of Pγ as depicted in Fig 1A
5. How is it thought that activated transducin α subunit binds to PDE6 ?
6. What was the source of PDE6 used in this study?
7. Draw a flow diagram indicating the steps involved in purification and characterisation of the Pγ truncation mutant proteins
8. Which Pγ construct or constructs in Fig 1B:
a. contains the intact α3 region only
b. contains both an intact α1 and an α2 region
c. is the shortest
9. From Fig 2, the authors conclude that the α3 region of Pγ is of central importance in stabilising the binding of Pγ to PDE6 active site, with only modest additional contributions from α1 and α2 regions. Explain in your own words how they reach this conclusion using only the data in Fig 2
10. From Fig 3, the authors conclude that the Pγ inhibition by C-terminal synthetic peptides is competitive with respect to cAMP but non-competitive with respect to cGMP. Explain in your own words how they reach this conclusion using only the data in Fig 3
11. Activated transducin α subunits were prepared in the presence of GTPγS. Briefly, what is GTPγS? (2 marks) What is its function in this context?
12. Where in its sequence is the "inducer region" of Pγ?
13. Why do the authors conclude that the "inducer region" may inhibit PDE6 by a mechanism different to the C terminal "blocker" mechanism?
14. Under what conditions did the authors observe inhibition by the "inducer region" to be less effective?
15. Summarise the mechanisms by which activated transducin is thought to relieve inhibition of PDE6 by Pγ
16. Explain the meaning of the asterisks in Figure 6
17. In your own words, summarise the main conclusions to emerge from the paper
18. In the final paragraph, the authors speculate that mutations in Pγ might give rise to defects in phototransduction, though this has not yet been observed in humans. Speculate on the effects you think a hypothetical mutation in one of the C-terminal regions of Pγ might have on the visual signal transduction pathway.