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? 1 2 3 O 11-cis

5. Where does it come from?

6. Your body turns  -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 J Biol Chem 1956 George Wald Nobel Prize 1967

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

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 1 2 3 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 Ground state Excited state

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

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

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

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:  max = 420, 535, 565 nm Rod:  max = 505 nm

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

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 + 11-cis retinal F261 Y261 + 11-cis retinal Y261

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

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

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

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

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

45. Opsin gene tandem arrays on X chromosome 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.

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

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

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

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

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

51. Deuteranomoly - green pigment shifted towards red  max = 420, 554, 565 nm 5% male 0.04% female

52. Mutations in human opsin genes Protanope Deuteranope Protanomalous Deuteranomalous

53. Color “blindness” DeficiencyMalesFemales Protanopia1%0.01% Deuteranopia1%0.01% Protanomoly1%0.01% Deuteranomoly5%0.4% Total (red-green)8%0.5% Tritanopia0.008%

54. Phylogenetics Compare sequences and determine the relatedness of things -Calculate % similarity of DNA or AA sequences Draw relatedness as a tree Human Mouse Bird Human Mouse Bird

55. Vertebrates Placental mammalsAmphibians MarsupialsBirds ReptilesCartilagenous fish Bony fishJawless fish

56. Vertebrate relationships and divergence times Kumar and Hedges 1998 Mammals, 100 MY Fish, 450 MY

57. Trees can also tell you about genes What organisms have the gene? Where did the gene come from? What happens to the gene once it’s there? Duplicate - tandem - mRNA can be inserted Lost

58. Line lengths are proportional to how different sequences are Human Chimp Dog Humans and chimps had a common ancestor 5-6 MYa so genes will be very similar Dogs and other mammals are about 100 MY apart so genes will be 20x more different from human as compared to human-chimp

59. Default expectation - if gene arose early in vertebrates, all species will have a copy and gene will be related in same way as organisms Dog Gene A Opossum Gene A Chicken Gene A Frog Gene A Zebrafish Gene A

60. Examine whether a gene exists in all organisms Dog Gene A Opossum Gene A Chicken Gene A Frog Gene A Zebrafish No A Gained

61. Examine whether a gene exists in all organisms Mouse Platypus Chicken Frog Pufferfish lost

62. What is happening? Gene duplication Human Gene A Chicken Gene A Frog Gene A Zebrafish Gene A1 Dog Gene A Zebrafish Gene A2

63. Human Chicken Frog Zebrafish Dog Human Chicken Frog Zebrafish Dog Lamprey Gene duplication GeneA2 GeneA1 Gene A

64. Human Frog Dog Chicken Frog Zebrafish Lamprey GeneA2 GeneA1 Gene A

65. Human Chicken Frog Zebrafish Dog Human Chicken Frog Zebrafish Dog Gene duplication and then losses Lamprey Gene A2 Gene A1

66. Opsin genes Opsin genes can duplicate Tandem duplication Chromosomal duplication Whole genome duplication Opsin genes can be lost Can reinsert from mRNA No introns

67. Opsin genes from: Lamprey (jawless vertebrate) Zebrafish Anole (reptile) Chicken (bird) Mouse Human

68. LWS RH2 SWS2 SWS1 RH1 What does this tree tell us?

69. Conclusions from opsin tree #1 5 opsin classes arose very early in vertebrates SWS1 - very short wavelength sensitive SWS2 - short wavelength sensitive RH2 - like rhopopsin but in cones LWS - long wavelength sensitive RH1 - rhodopsin rods cones

70. Range of cone visual pigment λ max SWS1 SWS2 RH2 LWS

71. Conclusion #2 Rod opsins evolved from cone opsins LWS SWS1 SWS2 RH2 RH1

72. LWS RH2 SWS2 SWS1 RH1 Mammalian genes

73. Conclusion #3 Mammals lost two of the opsin classes Mammals have LWS, SWS1 and RH1 Only 2 cone opsins (dichromat) Dogs, cats, mice, rats, horses, goats, pigs … Mammals went through “nocturnal period” during reign of dinosaurs

74. “Rugrats”

75. “Spike’s view”

76. Spike’s view?

77. LWS RH2 SWS2 SWS1 RH1 Human genes

78. Conclusion #4 Primates had a duplication of the LWS gene Went from dichromatic to trichromatic

79. Human green and red opsins are part of LWS class = M/LWS  max = 535, 565 nm

80. M/LWS opsin duplication on X chromosome Rhodopsin Chr 3 Blue opsin Chr 7 Red and green opsin - X chromosome

81. New world vs Old world primates X X

82. Why trichromacy? Why two ‘ LWS ’ cone types? Dichromacy with a single LWS and an SWS1 cone type gives no red-green discrimination. Jim Bowmaker

83. Trichromacy with two ‘ LWS ’ cone types and an SWS1 cone gives red-green discrimination. Ripe fruit and young, more reddish leaves can be detected against the dappled green foliage. Jim Bowmaker