Photoproteins are bioluminescent proteins found in light-emitting organs that produce light in proportion to the amount of protein present. There are various types of photoproteins that emit light in response to different stimuli like calcium ions, hydrogen peroxide, or ATP. Two major calcium-sensitive photoproteins are aequorin and obelin, which emit blue light when calcium is added. These proteins contain "EF-hand" motifs that allow them to tightly bind calcium ions. Aequorin luminesces when a conformational change occurs after calcium binds, causing a peroxide group to cyclize and emit a photon. Photoproteins can be used as calcium indicators in applications like neuronal cell biology.

1. 1

2. • Photoprotein:
- Bioluminescent proteins that occur in the light
organs of luminous organisms as the major
luminescent component
- Capable of emitting light in proportion to the
amount of protein
-More stable than its dissociated forms
2

3. Various Types of Photoproteins
•
•
•
•
•
•
•
•
•
Radiolarian
Ca2+ -sensitive photoprotein 2+ is
Emit blue light when Ca
Coelenterate Ca2+ -sensitive photoproteins
added, regardless of the presence
Ctenophore photosensitive oxygen
or absence of
Pholasin
Luciferin-luciferase reaction 2+
Emits light in the presence of Fe ,
Chaetopterus Emits light in the presence of
amolecular oxygen and superoxide
peroxide, and molecular oxygen.
Polynoidin
Emitted
radicals light in the presence of
Symplectin
monovalent cations
Emit light in the presence
LuminodesmusEmit a greenish-blue light inof
ATP, Mg2+ and molecular
Ophiopsila
the presence of H2O2
oxygen
3

4. Coelenterate
Aequorin from the jellyfish Aequorea
and
obelin from the hydroids Obelia
“precharged” bioluminescent proteins
+
Ca2+
Emit BLUE Light
4

5. Aequorin
“ Ca2+ -regulated photoproteins ”
• Members of the family of calcium regulated
proteins
• Calcium regulates the function of these proteins
but is not essential for it.
 Ca2+-free photoproteins emit a very low level of
light named “Ca2+ -independent bioluminescence”
, but the light intensity increases to 1-million fold
or even more after the addition of calcium.
5

6. Calcium-binding loops:
• The EF -hand proteins: have common calciumbinding HTH motifs consisting of two helices that
flank a canonical sequence loop region of 12
contiguous residues from which the oxygen
ligands for Ca2+ coordination are derived
• The calcium ion is coordinated in a pentagonal
bipyramidal array with anaverage of 2.4 Å
separation to oxygen atoms
• One or two water molecules are also involved in
ligating to calcium
6

7. Properties of EF-hand Calcium-Binding
Proteins
• the EF-hand motif always occurs in pairs. It is
assumed to be important for correct protein folding
and enhancing of the affinity of Ca2+ binding loops to
calcium.
• paired EF-hands display extensive hydrophobic
interhelical interactions and a short β-type interaction
between the two loops.
• Antiparallel β-type interaction is formed with the
main chain amide and carbonyl group of the adjacent
hydrophobic strand.
7

8. Structure of Aequorin
Globular protein
3 “EF-hand” domains
For binding to Ca2+
Peroxidized
coelenterazine as
functional group
8

9. 9

10. Luminescence Reaction
Apoaequorin + Peroxidazed Coelenterazine
(functional group)
Aequrin + Ca2+
Conformational change
Cyclization of peroxide of coalentrazine
Colentramide +Apoaequrin + CO2
10

11. Regeneration
Apoaequorin can be reconstituted into aequorin
by incubation with coelenterazine in the
presence of O2 and 2-mercaptoethanol, which
the role of the latter substance is to protect the
functional sulfhydryl groups of apoaequorin
during the regeneration reaction.
11

12. Luminescence Reaction
12

13. Berovin
Slightly asymmetric is
The protein globule
Two-domain
compact globular
formed byis stabilized
structure two cup-like
protein with its Ndomains contacting by
both by multiple inter
terminalhydrogen bonds
their rimsα-helix (1–
helical with a cavity
35) resting inhydrogen
in thethrough Each cup is
and center. the
side groove four -and
composed ofof theαbonds between N
protein.
helices A–D and E–H in
C -terminal domains
N-and С-terminal
domains
13

14. 12 residues of the I, III and IV Ca2+
binding loops coordinate calcium ions in apoberovin
I- Ca2+ binding loop
III -Ca2+ binding loop
IV -Ca2+ binding loop
14

15. • In Ca2+ binding loops I and III, the seventh
ligand comes from the oxygen atom of a water
molecule that is hydrogen bonded to another
ligand.
• In Ca2+ binding loop IV the water molecule is
missing; the side chain oxygen of Glu180
directly coordinates Ca2+ .
15

16. Active Site
••A more focused Obelin, three the
The binding site of view of
Hydroperoxy-coelenterazine binding
key residues of interest are
site reveals Hispink. These amongst on
highlighted in 22 binds to the O25
the hydroperoxy-coelenterazine. and
others form the binding pocket
• surrounding residues form the
Tyr190 which forms an hydrogen
bond to the N2 of network while also
hydrogen-bound His 175, of the
bindingcenter. hydroperoxy unit
active to the
bound to coelenterazine.
It has been observed that Ca2+ binding
sites with such type of coordination
possess a high affinity for Ca2+
16

17. Application of Photoproteins
17

18. Neuronal Cell Biology
• Importance of Ca2+ signaling
• Ca2+ indicators:
- Fluorescent dyes
- Genetically encoded fluorescent or
luminescent proteins:
Advantage
Disadvantage
18

19. advancements
• add target sequences to Ca2+ sensitive proteins
sequences, localize them in a specific cell
compartment where Ca2+ can be directly
monitored and acquire important information
on the organellar Ca2+ homeostasis.
• disadvantages like phototoxicity, photobleaching and autofluorescence.
19

20. • Ca2+ sensitive photoprotein aequorin
• bi-functional Ca2+ reporter gene” GFP–aequorin
• do not require to be excited by artificial
illumination
• Since it is not cell permeable, aequorin has
especially been used in giant cells, i.e. barnacle
muscle fibers, oocytes, or cells from heart, liver
and adrenal gland, where it was introduced by
permeabilization or microinjection procedures
• cDNA
20

21. 21

22. 22

23. Recombinantly expressed aequorin
Not exported or secreted or compartmentalized or
sequestered within cells
Permitting to detect Ca2+ changes over long periods.
Not toxic
Do not perturb cell functions or embryo development.
23

24. Measure cytosolic Ca2+ concentration
in several conditions:
1. To have a real
concentration values
quantification
of Ca2+
2. To monitor Ca2+ concentration in a subpopulation
of cells which express a particular repertoire of
proteins
3. To monitor cytosolic Ca2+ variations in the range
of micromolar values that may occur for example
in excitable cells such as neurons
24

25. Recombinant aequorin
• Modify its Ca2+ sensitivity by:
1. The mutation of amino acid residues in
the “EF-hand” domains to reduce the
affinity of aequorin for Ca2+
2. The use of surrogate cations which elicit a
slower rate of photoprotein consumption
3. The employment of modified synthetic
prosthetic groups which reduce the affinity of
aequorin for Ca2+
25

26. Thank you
26