Lecture #5

Vertebrate visual pigments2/7/13

HW #3

• There are two things on the assignment page: Assign#3.pdf which has the homework

problemsHumanGreenRedCones.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

Today’s topics

• Visual pigments and opsin genes

• Opsin gene classes and diversity in vertebrates

• Primates: di- and trichromacy

What absorbs light in a visual pigment?

12

3

O11-cis

Where does it come from?

Where does it come from?

Your body turns β-carotene into vitamin A

All trans-retinOL

Where does it come from?

Vitamin A is converted to 11-cis retinal (visual cycle)

Absorption spectrum of 11-cis retinal

J Biol Chem 1956

George WaldNobel Prize 1967

Absorption spectrum of 11-cis retinal

378 nm

11-cis retinal absorption

11-cis retinal vs human visual pigments - opsin shift

11-cis S M L

What is a visual pigment?

• Opsin protein surrounding and bound to 11-cis retinal

• Transmembrane proteinContained in the membrane

• G protein coupled receptorTurns on a G protein

Membrane holds the visual pigment

Rods have discsCones have continuous membrane

Opsin protein is threaded through the membrane

80% of protein in outer segment is rhodopsin

Rhodopsin crystal structure

Visual pigment =opsin + retinal

11-cis retinal

membrane

In rod, visual pigment is called rhodopsin

Retinal is in binding pocket of opsin protein

Chang et al. 1995

11-cis bond isomerizes to form all trans

Chang et al. 1995

Light causes isomerization

11-cis retinal + photon = all trans retinal

12

3

Light

Absorbing light

Excited state - stays as metaII

Meta II actually is what can activate the G protein

Excited state - stays as metaII

Eventually the excited state decays

All trans retinal dissociates, leaving opsin

Opsin recombines with new 11cis retinal

Electronic energy levels of visual pigment molecule

Ground state

Excited state

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

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

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

Opsin is bound to and surrounds 11-cis retinal

Chang et al. 1995

How do we get one rod and three cone visual pigments?

Cones: λmax = 420, 535, 565 nm Rod: λmax = 505 nm

Put a different opsin protein in each cone type

Webvision

Blue cone - blue opsin

Green cone - green opsin

Red cone - red opsin

Rod - rhodopsin

Blue opsin versus green opsin

Human rhodopsin sequence

Human Rh

sequence

Nathans et al 1986

Humans have 3 cone opsin genes

Blue opsin - 5 exons

Green and red - 6 exons

Sequences for human green and red opsin genes are VERY similar

HumanGreen MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60HumanRed MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60 ************************************************************

HumanGreen IFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFV 120HumanRed IFVVTASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISIVNQVSGYFV 120 **** *********************************************:**** ****

HumanGreen LGHPMCVLEGYTVSLCGITGLWSLAIISWERWMVVCKPFGNVRFDAKLAIVGIAFSWIWA 180HumanRed LGHPMCVLEGYTVSLCGITGLWSLAIISWERWLVVCKPFGNVRFDAKLAIVGIAFSWIWS 180 ********************************:**************************:

HumanGreen AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYL 240HumanRed AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCIIPLAIIMLCYL 240 ************************************************* **:**:****

HumanGreen QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMVLAFCFCWGPYAFFACFAAANPGYPFH 300HumanRed QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMIFAYCVCWGPYTFFACFAAANPGYAFH 300 *********************************::*:*.*****:************.**

HumanGreen PLMAALPAFFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360HumanRed PLMAALPAYFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360 ********:***************************************************

HumanGreen VSPA 364HumanRed VSPA 364 ****

Differ by 15 AA

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

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

You can mutate one amino acid and see how absorption peak

shifts

F261 F261 F261

+11-cis retinal

F261

Y261 Y261 Y261

+11-cis retinal

Y261

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

nm

Human red and green opsins

535 nm

565 nm

A

S

A

A164S=+2 nm

Y

F

T

F261Y=+10 nmA269T=+14 nm

These 3 AA explain most of the shift between red and green opsin genes

Location of human opsin genes

RhodopsinChr 3

Blue opsinChr 7

Exon

Red and green opsin - X chromosome

Normal DNA recombination

Switches genes from one chromosome to the other

Leads to new gene combinations

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

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.

Misaligned recombination

If recombination happens within gene, get chimeraIntermediate phenotype which results in color blindness

Opsin genes on X chromosome

Human red and green opsins

535 nm

565 nm

A

S

A

A164S=+2 nm

Y

F

T

F261Y=+10 nmA269T=+14 nm

554 nm

Chimera has intermediate peak wavelengthA YT

Protanope - no red cones1% males 0.01% females

λmax = 420, 535nm

Deuteranope - no green cones1% males 0.01% females

λmax = 420, 565 nm

Protanomoly - red pigment shifted towards green

λmax = 420, 535, 550 nm

1% male 0.01% female

Deuteranomoly - green pigment shifted towards red

λmax = 420, 554, 565 nm

5% male 0.04% female

Mutations in human opsin genes

Protanope

Deuteranope

Protanomalous

Deuteranomalous

Color “blindness”

Deficiency Males Females

Protanopia 1% 0.01%

Deuteranopia 1% 0.01%

Protanomoly 1% 0.01%

Deuteranomoly 5% 0.4%

Total (red-green) 8% 0.5%

Tritanopia 0.008% 0.008%

Phylogenetics

• Compare sequences and determine the relatedness of things- Calculate % similarity of DNA or AA

sequences

• Draw relatedness as a treeHuman

Mouse

Bird

Human

Mouse

Bird

Vertebrates

• Placental mammals Amphibians• Marsupials Birds• Reptiles Cartilagenous

fish• Bony fish Jawless fish

Vertebrate relationships and divergence times

Kumar and Hedges 1998

Mammals, 100 MY

Fish, 450 MY

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 insertedLost

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

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 AChicken Gene A

Frog Gene A

Zebrafish Gene A

Examine whether a gene exists in all organisms

Dog Gene A

Opossum Gene AChicken Gene A

Frog Gene A

Zebrafish No A

Gained

Examine whether a gene exists in all organisms

Mouse

Platypus

Chicken

Frog

Pufferfish

lost

What is happening?

• Gene duplication

Human Gene A

Chicken Gene A

Frog Gene A

Zebrafish Gene A1

Dog Gene A

Zebrafish Gene A2

Human

Chicken

Frog

Zebrafish

Dog

Human

Chicken

Frog

Zebrafish

Dog

Lamprey

Gene duplication

GeneA2

GeneA1

Gene A

Human

Frog

Dog

Chicken

Frog

Zebrafish

Lamprey

GeneA2

GeneA1

Gene A

Human

Chicken

Frog

Zebrafish

Dog

Human

Chicken

Frog

Zebrafish

Dog

Gene duplication and then losses

Lamprey

Gene A2

Gene A1

Opsin genes

• Opsin genes can duplicateTandem duplicationChromosomal duplicationWhole genome duplication

• Opsin genes can be lost

• Can reinsert from mRNANo introns

Opsin genes from:

• Lamprey (jawless vertebrate)• Zebrafish• Anole (reptile)• Chicken (bird)• Mouse• Human

LWS

RH2

SWS2

SWS1

RH1

What does this tree tell us?

Conclusions from opsin tree #1

• 5 opsin classes arose very early in vertebratesSWS1 - very short wavelength sensitiveSWS2 - short wavelength sensitiveRH2 - like rhopopsin but in conesLWS - long wavelength sensitiveRH1 - rhodopsin

rods

cones

Range of cone visual pigment λmax

SWS1SWS2

RH2LWS

Conclusion #2

• Rod opsins evolved from cone opsins

LWS

SWS1

SWS2

RH2

RH1

LWS

RH2

SWS2

SWS1

RH1

Mammalian genes

Conclusion #3

• Mammals lost two of the opsin classesMammals 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

“Rugrats”

“Spike’s view”

Spike’s view?

LWS

RH2

SWS2

SWS1

RH1

Human genes

Conclusion #4

• Primates had a duplication of the LWS gene

• Went from dichromatic to trichromatic

Human green and red opsins are part of LWS class = M/LWS

λmax = 535, 565 nm

M/LWS opsin duplication on X chromosome

RhodopsinChr 3

Blue opsinChr 7

Red and green opsin - X chromosome

New world vs Old world primates

X

X

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

Jim Bowmaker

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