1. Lecture #5 Vertebrate visual pigments 2/7/13

2. HW #3 • There are two things on the assignment page: • Assign#3.pdf which has the homework problems • HumanGreenRedCones.xlsx which is a spreadsheet you can use for one of the problems • I think I turned on online submissions but you don’t have to use that

3. Today’s topics • Visual pigments and opsin genes • Opsin gene classes and diversity in vertebrates • Primates: di- and trichromacy

4. What absorbs light in a visual pigment? 3 1 2 O 11-cis

5. Where does it come from?

6. Where does it come from? Your body turns β-carotene into vitamin A All trans-retinOL

7. Where does it come from? Vitamin A is converted to 11-cis retinal (visual cycle)

8. Absorption spectrum of 11-cis retinal George Wald Nobel Prize 1967 J Biol Chem 1956

9. Absorption spectrum of 11-cis retinal 378 nm

10. 11-cis retinal absorption

11. 11-cis retinal vs human visual pigments - opsin shift 11-cis S M L

12. What is a visual pigment? • Opsin protein surrounding and bound to 11-cis retinal • Transmembrane protein • Contained in the membrane • G protein coupled receptor • Turns on a G protein

13. Membrane holds the visual pigment Rods have discs Cones have continuous membrane

14. Opsin protein is threaded through the membrane 80% of protein in outer segment is rhodopsin

15. Rhodopsin crystal structure

16. membrane Visual pigment =opsin + retinal In rod, visual pigment is called rhodopsin 11-cis retinal

17. Retinal is in binding pocket of opsin protein Chang et al. 1995

19. 11-cis bond isomerizes to form all trans Chang et al. 1995

20. Light causes isomerization 11-cis retinal + photon = all trans retinal 3 1 2 Light

21. Absorbing light

22. Excited state - stays as metaII Meta II actually is what can activate the G protein

23. Excited state - stays as metaII Eventually the excited state decays All trans retinal dissociates, leaving opsin Opsin recombines with new 11cis retinal

24. Electronic energy levels of visual pigment molecule Excited state Ground state

25. Electronic energy levels of visual pigment molecule Excited state E = hc/λ Visual pigment absorbs light at wavelengths which can excite electrons to upper excited state light Ground state

26. Opsin interacts with retinal to make ground and excited states closer together Excited state E = hc/λ light Energy needed to excite electrons goes down Absorption is at longer wavelength Ground state

27. Opsin interacts with retinal to make ground and excited states farther apart Excited state E =hc/λ light Energy needed to excite electrons goes up Absorption is at shorter wavelength Ground state

28. Opsin is bound to and surrounds 11-cis retinal Chang et al. 1995

29. How do we get one rod and three cone visual pigments? Cones: λmax = 420, 535, 565 nm Rod: λmax = 505 nm

30. Put a different opsin protein in each cone type Blue cone - blue opsin Green cone - green opsin Red cone - red opsin Rod - rhodopsin Webvision

31. Blue opsin versus green opsin

32. Human rhodopsin sequence

33. Human Rh sequence

34. Nathans et al 1986

35. Humans have 3 cone opsin genes Blue opsin - 5 exons Green and red - 6 exons

36. Sequences for human green and red opsin genes are VERY similar HumanGreen MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60 HumanRed MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60 ************************************************************ HumanGreen IFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFV 120 HumanRed IFVVTASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISIVNQVSGYFV 120 **** *********************************************:**** **** HumanGreen LGHPMCVLEGYTVSLCGITGLWSLAIISWERWMVVCKPFGNVRFDAKLAIVGIAFSWIWA 180 HumanRed LGHPMCVLEGYTVSLCGITGLWSLAIISWERWLVVCKPFGNVRFDAKLAIVGIAFSWIWS 180 ********************************:**************************: HumanGreen AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYL 240 HumanRed AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCIIPLAIIMLCYL 240 ************************************************* **:**:**** HumanGreen QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMVLAFCFCWGPYAFFACFAAANPGYPFH 300 HumanRed QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMIFAYCVCWGPYTFFACFAAANPGYAFH 300 *********************************::*:*.*****:************.** HumanGreen PLMAALPAFFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360 HumanRed PLMAALPAYFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360 ********:*************************************************** HumanGreen VSPA 364 HumanRed VSPA 364 **** Differ by 15 AA

37. Why opsins are so cool • You can grow cells that express ANY opsin protein you want • You can add 11-cis retinal and purify the protein + 11-cis retinal

38. Why opsins are so cool • You can grow cells that express ANY opsin protein you want • You can add 11-cis retinal and purify the protein • You can measure the absorption spectrum for that visual pigment + 11-cis retinal

39. You can mutate one amino acid and see how absorption peak shifts F261 Y261 F261 F261 Y261 Y261 + 11-cis retinal + 11-cis retinal F261 Y261

40. Changing site 261 from F to Y shifts absorption peak by +10 nm

41. A F A 535 nm 565 nm S Y T F261Y=+10 nm A269T=+14 nm A164S=+2 nm Human red and green opsins These 3 AA explain most of the shift between red and green opsin genes

42. Location of human opsin genes Exon Rhodopsin Chr 3 Blue opsin Chr 7 Red and green opsin - X chromosome

43. Normal DNA recombination Switches genes from one chromosome to the other Leads to new gene combinations

44. If chromosomes misalign, recombination leads to gain in genes on one chromosome and loss of genes on the other. Tandem arrays of genes Mismatched recombination

45. Humans differ in how many copies they have of green gene. Only first 2 genes are expressed so it doesn’t matter if there are more green genes. They are just along for ride. Opsin gene tandem arrays on X chromosome

46. If recombination happens within gene, get chimera Intermediate phenotype which results in color blindness Misaligned recombination Opsin genes on X chromosome

47. 535 nm 565 nm 554 nm Human red and green opsins A F A F261Y=+10 nm A269T=+14 nm A164S=+2 nm S Y T Chimera has intermediate peak wavelength A YT

48. Protanope - no red cones 1% males 0.01% females λmax = 420, 535nm

49. Deuteranope - no green cones 1% males 0.01% females λmax = 420, 565 nm

50. Protanomoly - red pigment shifted towards green 1% male 0.01% female λmax = 420, 535, 550 nm