Chemistry of photoreceptors
Contents
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Introduction
Red/Far red photoreceptors
Blue light photoreceptors
Blue/UV-A light photoreceptors
Green Fluorescent protein(GFP)
Introduction
• Light is vital for a plant’s life - regulates plant growth and
development.
• Three major categories of photoresponses
• Phototropism(Photosynthesis)
• Photoperiodism(seed germination)
• Photomorphogenesis(synthesis of chlorophyll)
Ref: Phytochrome and photomorphogenesis, Harry Smith
•
What are photopigments
?
Organic molecule which absorbs in the near UV-Visible range
and upon absorption of the photon initiates a chemical reaction.
Criteria
• Absorption spectrum of the pigment should overlap with the
wavelengths that are abundantly present in sunlight.
• The pigment must have high extinction coefficient.
• The excited state of the photopigment must have long lifetime.
Ref: Light signals and the growth and development of plants - a gentle introduction, Pedro J. Aphalo
Classification of photopigments
Two types of photopigments
Mass pigments
• Absorbs large
fraction of light.
• Harvest energy
(egs). Chlorophyll,
flavonoids etc
Sensor pigments
• Absorbs small
fraction of light.
• Adjusts behaviour
of plants.
• red/far red, UV-A
UV-B photoreceptors
Ref: Aziz Sancar,Annu. Rev. Biochem., 2000(69), 31-67
Types of photoreceptors
1. Rhodopsins(visible)
2. Phytochromes(red/far red)
3. Xanthopsins(blue)
4. Cryptochromes(Blue/UV-A)
“Primary photochemistry of
photoreceptors activation is
cis-trans isomerisation of the
chromophore”
Photoreceptors and their functions
• Rhodopsins - Black & white and colour vision in higher animals
• Light harvesting complex(LHC) - Photosynthesis in plants.
• Phytochrome - Photomorphogenesis in plants
• Cryptochrome - Regulation of circadian rhythm in living
organism.
• Photolyase - Repair of DNA damage in drosophila, xenopus etc.
Classification of photoreceptors
CHROMOPHORES
Classes
PHOTOSENSORS PHOTOCHEMISTRY
FAMILY
example
structural unit
R1
N
Tetrapyrroles phytochromobilin
R2
Phytochromes
trans
Cis
Rhodopsins
trans
Cis
Xanthopsins
trans
Cis
N
O
Polyenes
retinal
Coumaric acid
13
11
12
14
N
H
O
-O
S-
Aromatics
R
Flavin
N
N
O
Cryptochromes
electron transfer
N
N
O
Ref: Klass J. Hellingwerf, J. Photchem. Photobiol. B, 2000(54), 94-102
Phytochrome
Red/far red photoreceptor
Introduction
• Phytochrome was discovered by Borthwick and Hendrick while
trying to understand the photoperiodic control of flower
formation and light regulation of seed germination.
• Phytochrome is the best known light receptor in all higher plants,
algae, mosses etc.
• Phytochrome absorbs visible light both in blue and red regions.
• Energy of the absorbed light is then converted to signals which
exert photomorphogenic controls (i.e., control of plant growth).
• Phytochromes are responsible for controlling seed germination,
stem elongation, leaf expansion, synthesis of chlorophyll etc.
Structure of phytochrome
• Phytochrome is a protein with molecular weight of 124 kDa
• Phytochrome molecule has a tetrapyrrole chromophore absorbing
in the red region.
• The chromophore is bound to sulfur of cystein-321.
• The structure of the chromphore(tetrapyrrole) was confirmed
by oxidative degradation by chromic acid.
• Phytochrome(P) can exist in two relatively stable forms
1. Physiologically inactive form(Pr)-red absorbing form
2. Physiologically active form(Pfr)-far red absorbing form
Photochemistry of Phytochrome chromopho
660 nm
Pfr
Pr
Physiological reaction
730 nm
Ref: W. Rudiger, F. Thummler., Angew. Chem. Intl. Ed., 1991(30), 1216-1228
Mechanism for the conversion of Pr to Pfr
• Z, E isomerisation of the chromophore
C16
C5
C15
C10
red light
far red light
• Pr chromophore - 5Z, 10Z, 15Z
Pfr chromophore - 5Z, 10Z, 15E
Evolution of Phytochrome function
• Phytochrome has five genes (phyA, phyB, phyC, phyD, phyE)
• Phytochrome regulates almost all phases of plant development.
“Single input/multiple ouput” sensory systems
How does phytochrome work ?
• Sunlight(660 nm) converts Pr to Pfr, the Pfr moves from the
cytoplasm into the nucleus.
• It binds then to a protein called PIF3(“phytochrome-interacting
factor 3”) which is a helix-loop-helix protein.
• The complex of two then binds to the promoters containing the
sequence CACGTG, GTGCAC.
• These promoters are found in genes that themselves encode
other transcription factors and initiate transcription.
• Exposure to far red light converts the Pfr back to Pr form and PIF3
dissociates from Pfr.
Photoactive Yellow Protein(PYP)
Blue light photoreceptor
Introduction
• PYP was discovered almost 20 years ago in Ectothioshodospira
Halophila.
• PYP belongs to Xanthopsins family - a blue light photoreceptor
protein.
• PYP is the biopigment responsible for photophotactic activity of
the purple phototropic bacteria.
• Purple phototropic bacteria(H. halophilia) is not immune to UV
light. It is attracted by infra red light(for photosynthesis) but moves
away from blue light.
Photoresponses of R. Centenum.
Positive movement towards infrared light
Negative movement towards visible light.
Accumulation pattern of cells of E.halophilia in
response to blue light illumination.
Structure of Photoactive yellow
protein
• 125 amino acid residues with molecular weight of ~ 14 KDa
-O
O
S
Protein
Chromophore structure
p-hydroxy thiocinnamate
Structure of PYP from Halorhodospira halophila
Ref: K. J. Hellingwerf, J. Hendriks, T. Gensch, J. Phys. Chem. A, 2003(107), 1082-1094
• The chromophore is linked through a thiol ester linkage to Cys69.
• Negative charge is stabilized via a hydrogen bonding network
with residues Try42, Glu46, Thr50.
• UV/Vis absorption(solid line) and fluorescence emission spectrum
λmax = 446nm
φfluo = 0.002
The PYP Photocycle
• PYP* - excited state species
• pR - redshifted photoproduct
• pB - blueshifted photoproduct
• More detailed analysis of low temperature spectroscopic
experiments revealed branched pathways.
A Comprehensive PYP photocycle
• Intermediates of photocycle
of PYP were determined at
- 12°C.
• At very low temperatures(-120 º C)
another intermediate PYPBL was
found.
• Analysis of crystal structures of
photoproducts indicated that the
key feature is the flipping of the
carbonyl group of the thioester
linkage.
• Signalling state is pB.
• pB interacts with transducer protein.
Cryptochromes
Blue light/UV A radiation
photoreceptors
Introduction
• Cryptochromes are receptors of blue light and UV A radiation.
• Blue light responses are present in all species tested from
bacteria to plants and animals.
• Various responses observed are photoactivation in bacteria,
phototropism, photomorphogenesis in plants, circadian rhythm
in fungi and drosophila.
Types of Cryptochromes
1. Photolyase - involved in DNA repair mechanism Drosophila, virus.
2. HY, phototropin - regulates phototropism in plants
3. CRY1, CRY2 - mediate circadian clock in humans.
Biological clocks
• Most of our activities follows a set of pattern - “Biological
rhythm”.
• Biological rhythm is an expression of internal clock(biological
clock).
• Circadian rhythm - biological functions with periodicity
of approximately 24 hours.
• Circadian rhythms are observed in most living organisms
ranging from cyanobacteria to humans to plants.
• hCRY1 and hCRY2 are the proteins which mediate the
biological clock.
• hCRY1 is 586 amino acid long 66 kDa protein.
hCRY2 is 593 amino acid long 67 kDa protein.
Photolyase
• Photolyase is a 55 to 64 KDa protein.
• Repairs UV induced DNA damage in a reaction dependent on
near UV to blue light(350-450 nm).
Ref: H. Komori, R. Masui, S. Kuramitsu, S. Yokoyama, T. Shibata, Y. Inoue,
K. Miki, PNAS, 2001(98), 13560-13565
DNA damage by UV light
• Absorption of UV light by DNA results in the formation of
triplet state of thymine.
• Thymine can return to ground state or can undergo a pericyclic
reaction(2+2 cycloaddition) with adjacent pyrimidine ring of
thymine or cytosine.
O
O
NH
O
OR
N
O
UV
O
O
O
P
NH
N
H
O
O
O
O
HN
NH
O
N
O
OR
OR
O-
photolyase
N
O
O
O
near UV-visible
H
O
P
O-
OR
O
Photoreactivation
• Damaged DNA can be repaired by photolyases activated by
blue/UV A light.
• Chromophore in photolyase responsible for DNA repair.
a) Flavin adenine dinucleotide(FAD) in their anionic form
b) Methenyl tetrahydrofolate(MTHF) or 8-Hydroxy 5 deaza
riboflavin.
Electronic energy transfer
• The two chromophores folate(MTHF) and flavin(FADH-) have
different characteristics.
Folate(MTHF): ε = 25000 M-1cm-1
Flavin(FADH-): ε = 5000 M-1cm-1
• Dependence of light penetration into the cells shows that folate
excels flavin as a light absorber.
• Energy transfer from folate(donor) to acceptor(flavin) occurs
by Forster type energy transfer.
kET ∝ k2J/R6
k2 = orientation of interacting dipoles
J = spectral overlap
R= distance between the dipoles
Mechanism
• The photoreactivation enzyme(photolyase) binds to pyrimidine
dimers in the DNA(Kasso = 2.6 x 108 M-1).
• Upon exposure to light, MTHF absorbs a photon(photoantenna)
and transfers excitation energy to flavin.
• Flavin transfers an e- to the DNA photoproduct: the cyclobutane
ring of the pyrimidine is broken to generate two pyrimidine
rings.
• Back electron transfer restores the FADH- and enzyme dissociates
from DNA.
• Quantum yield of the reaction is found to be 0.5-0.9.
Ref: J. E. Hearst, Science, 1995(268), 1858-1859
Green Flurorescent Protein
• GFP was first found in a jellyfish named Aequorea which
fluoresced green when irradiated with UV light.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Chromophore structure
p-hydroxy benzylidene
• Chromophore is buried under compact, rigid protective
structure - highly stable and has high quantum yield.
• Isolated chromophore is not fluorescent whereas the folded
protein is fluorescent in nature.
• Chromophore formation - Autocatalytic postransational cyclisation.
• Depending on the chromophore, GFP can be classified into seven
classes.
Mechanism of chromophore formation
• Chromophore formation is the bottleneck of protein folding.
Mechanism of fluorescence
• GFP has two absorption peaks - at 398 nm and 475 nm.
• Emission at 503 to 509 nm when excited at 398 nm and emits at
482 nm when excited at 475 nm.
• Two absorption states:
Chromophore in phenolic neutral form(A state)
Chromophore in phenolate anionic form(B state)
• Excited state proton transfer from
chromophore to Glu222.
Applications of GFP
• The applications of GFP are as Fusion tags, Reporter gene,
biosensors, photobleaching, FRET etc.
1. Fusion Tag
• Fusion between cloned gene and GFP can be used to visualize
dynamic cellular events.
2. Reporter Gene
• Gene expression can be monitored by using a GFP gene that is
under the control of the promoter of interest.
• The change in the intensity of fluorescence of GFP directly
indicates the level of gene expression.
3. Fluorescence Resonance Energy Transfer
• Folding and Unfolding dynamics of protein can be studied using
GFP.
CFP, YFP is connected to
metal lathionein(Zn)
Add EDTA
Unfolding - Zn binds
with EDTA
(F535nm/F480nm) decreases
4. Biosensors and Bioelectronics
• Genetically modified plants are being used as biosensors.
• Biosensors responsive to copper and TNT has been designed.
• Concept of photobleaching is now been exploited for
bioelectronics and memory storage devices.
5. How does a non-scientist looks at GFP ?
GFP-bunny
GFP-monkey
Conclusion
• Photochemistry plays a important role in everyday life starting
from control of plant growth, photosynthesis in plants,
black and white and colour vision in higher animals.