Lecture 5

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Lecture #5
Vertebrate visual pigments
2/7/13
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
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?
3
1
2
O
11-cis
Where does it come from?
Where does it come from?
Your body turns -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
George Wald
Nobel Prize 1967
J Biol Chem 1956
Absorption spectrum of 11-cis retinal
378 nm
11-cis retinal absorption
1.20
Relative absorption
1.00
0.80
0.60
0.40
0.20
0.00
300
350
400
450
500
550
Wavelength (nm)
600
650
700
11-cis retinal vs human visual
pigments - opsin shift
1.20
Relative absorption
1.00
11-cis
S
M
L
0.80
0.60
0.40
0.20
0.00
300
350
400
450
500
550
Wavelength (nm)
600
650
700
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
Membrane holds the visual pigment
Rods have discs
Cones 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
In rod, visual
pigment is called
rhodopsin
membrane
11-cis retinal
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
3
1
2
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
Electronic energy levels of visual
pigment molecule
Excited
state
Ground
state
Electronic energy levels of visual
pigment molecule
Excited
state
E = hc/λ
light
Ground
state
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
Excited
state
E = hc/λ
light
Energy needed to
excite electrons goes
down
Ground
state
Absorption is at
longer wavelength
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
Ground
state
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:  max = 420, 535, 565 nm
Rod:  max = 505 nm
1.20
Relative absorption
1.00
0.80
0.60
0.40
0.20
0.00
300
350
400
450
500
550
Wavelength (nm)
600
650
700
Put a different opsin protein in each
cone type
Blue cone - blue opsin
Green cone - green opsin
Red cone - red opsin
Rod - rhodopsin
Webvision
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
HumanRed
MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60
MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM 60
************************************************************
HumanGreen
HumanRed
IFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFV 120
IFVVTASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISIVNQVSGYFV 120
**** *********************************************:**** ****
HumanGreen
HumanRed
LGHPMCVLEGYTVSLCGITGLWSLAIISWERWMVVCKPFGNVRFDAKLAIVGIAFSWIWA 180
LGHPMCVLEGYTVSLCGITGLWSLAIISWERWLVVCKPFGNVRFDAKLAIVGIAFSWIWS 180
********************************:**************************:
HumanGreen
HumanRed
AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYL 240
AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCIIPLAIIMLCYL 240
************************************************* **:**:****
HumanGreen
HumanRed
QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMVLAFCFCWGPYAFFACFAAANPGYPFH 300
QVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMIFAYCVCWGPYTFFACFAAANPGYAFH 300
*********************************::*:*.*****:************.**
HumanGreen
HumanRed
PLMAALPAFFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360
PLMAALPAYFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSS 360
********:***************************************************
HumanGreen
HumanRed
VSPA 364
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
+
11-cis retinal
LWS cone
1.0
relative absorbance
• 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
0.8
0.6
0.4
0.2
0.0
300
400
500
wavelength, nm
600
700
You can mutate one amino acid and
see how absorption peak shifts
F261
F261
F261
Y261
Y261
Y261
+
11-cis retinal
+
11-cis retinal
F261
Y261
Changing site 261 from F to Y shifts
absorption peak by +10 nm
1.2
Relative absorption
1
0.8
0.6
F261
Y261
0.4
0.2
0
300
350
400
450
500
550
Wavelength (nm)
600
650
700
Human red and green opsins
A
FA
535 nm
565 nm
S
A164S=+2 nm
YT
F261Y=+10 nm
A269T=+14 nm
These 3 AA explain most of the shift between red and green opsin genes
Location of human opsin genes
Exon
Rhodopsin
Chr 3
Blue opsin
Chr 7
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
Opsin genes on X chromosome
If recombination happens within gene, get chimera
Intermediate phenotype which results in color blindness
Human red and green opsins
A
FA
535 nm
F261Y=+10 nm
A269T=+14 nm
A164S=+2 nm
565 nm
S
YT
Chimera has intermediate peak wavelength
A
YT
554 nm
Protanope - no red cones
1% males
0.01% females
 max = 420, 535nm
Deuteranope - no green cones
1% males
0.01% females
 max = 420, 565 nm
Protanomoly - red pigment shifted
towards green
1% male 0.01% female
 max = 420, 535, 550 nm
Deuteranomoly - green pigment
shifted towards red
5% male 0.04% female
 max = 420, 554, 565 nm
Mutations in human opsin genes
Deuteranope
Protanope
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 tree
Human
Human
Mouse
Mouse
Bird
Bird
Vertebrates
•
•
•
•
Placental mammals
Marsupials
Reptiles
Bony fish
Amphibians
Birds
Cartilagenous fish
Jawless fish
Vertebrate relationships and
divergence times
Human
92
71
Cow
Mammals, 100 MY
41 Mouse
Rat
276
Reptiles
310
Birds
360
Amphibians
Tilapia
120
200
Fugu
250
Cartilagenous fish
564
500
400
Fish, 450 MY
Zebrafish
528
600
Bony
fish
Agnatha
300
Time (Myr)
200
100
0
Kumar and
Hedges 1998
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
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 A
Chicken Gene A
Frog Gene A
Zebrafish Gene A
Examine whether a gene exists in all
organisms
Dog Gene A
Gained
Opossum Gene A
Chicken Gene A
Frog Gene A
Zebrafish No A
Examine whether a gene exists in all
organisms
Mouse
Platypus
lost
Chicken
Frog
Pufferfish
What is happening?
• Gene duplication
Dog Gene A
Human Gene A
Chicken Gene A
Frog Gene A
Zebrafish Gene A1
Zebrafish Gene A2
Gene duplication
Dog
Human
Chicken
GeneA2
Frog
Zebrafish
Dog
Human
Chicken
GeneA1
Frog
Zebrafish
Gene A
Lamprey
Dog
GeneA2
Human
Frog
Chicken
GeneA1
Frog
Zebrafish
Gene A
Lamprey
Gene duplication and then losses
Dog
Human
Chicken
Gene A2
Frog
Zebrafish
Dog
Human
Chicken
Gene A1
Frog
Zebrafish
Lamprey
Opsin genes
• Opsin genes can duplicate
Tandem duplication
Chromosomal duplication
Whole genome duplication
• Opsin genes can be lost
• Can reinsert from mRNA
No introns
Opsin genes from:
•
•
•
•
•
•
Lamprey (jawless vertebrate)
Zebrafish
Anole (reptile)
Chicken (bird)
Mouse
Human
SWS2
RH2
RH1
SWS1
LWS
What does this tree
tell us?
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
cones
rods
Range of cone visual pigment λmax
SWS2
SWS1
LWS
RH2
Conclusion #2
• Rod opsins evolved from cone opsins
RH1
RH2
SWS2
SWS1
LWS
SWS2
RH2
RH1
SWS1
LWS
Mammalian genes
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
“Rugrats”
“Spike’s view”
Spike’s view?
SWS2
RH2
RH1
SWS1
LWS
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
 max = 535, 565 nm
M/LWS opsin duplication on X
chromosome
Rhodopsin
Chr 3
Blue opsin
Chr 7
Red and green opsin - X chromosome
X
New world vs Old world primates
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

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