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Photoproteins

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• 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

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Various Types of Photoproteins

• Radiolarian• Coelenterate• Ctenophore• Pholasin• Chaetopterus• Polynoidin• Symplectin• Luminodesmus• Ophiopsila

Ca2+ -sensitive photoproteinEmit blue light when Ca2+ is added, regardless of the presence or absence of oxygen

Ca2+ -sensitive photoproteinsphotosensitiveLuciferin-luciferase reaction

Emits light in the presence of Fe2+ , a peroxide, and molecular oxygen.Emits light in the presence of molecular oxygen and superoxide radicalsEmitted light in the presence of monovalent cations

Emit light in the presence of ATP, Mg2+ and molecular oxygen

Emit a greenish-blue light in the presence of H2O2

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Coelenterate

Aequorin from the jellyfish Aequorea

and

obelin from the hydroids Obelia

“precharged” bioluminescent proteins

+

Ca2+

Emit BLUE Light

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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.

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Calcium-binding loops:

• The EF -hand proteins: have common calcium-binding 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

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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.

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Structure of Aequorin

Globular protein

3 “EF-hand” domainsFor binding to Ca2+

Peroxidized coelenterazine as functional group

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Luminescence Reaction

Apoaequorin + Peroxidazed Coelenterazine

(functional group)

Aequrin + Ca2+

Conformational change

Cyclization of peroxide of coalentrazine

Colentramide +Apoaequrin + CO2

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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.

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Luminescence Reaction

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Slightly asymmetric compact globular protein with its N-terminal α-helix (1–35) resting in the side groove of the protein.

The protein globule is formed by two cup-like domains contacting by their rims with a cavity in the center. Each cup iscomposed of four α- helices A–D and E–H in N-and С-terminal domains

Two-domainstructure is stabilized both by multiple inter helical hydrogen bonds and through hydrogen bonds between N -and C -terminal domains

Berovin

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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

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• 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+ .

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• A more focused view of the Hydroperoxy-coelenterazine binding site reveals His 22 binds to the O25 on the hydroperoxy-coelenterazine. • Tyr190 which forms an hydrogen bond to the N2 of His 175, while also binding to the hydroperoxy unitbound to coelenterazine.

• The binding site of Obelin, three key residues of interest are highlighted in pink. These amongstothers form the binding pocket and surrounding residues form the hydrogen-bound network of the active center.

Active Site

It has been observed that Ca2+ binding sites with such type of coordination possess a high affinity for Ca2+

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Application of Photoproteins

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Neuronal Cell Biology

• Importance of Ca2+ signaling• Ca2+ indicators:

- Fluorescent dyes

- Genetically encoded fluorescent or luminescent proteins:

Advantage

Disadvantage

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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, photo-bleaching and autofluorescence.

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• 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

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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.

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1. To have a real quantification of Ca2+ concentration values

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

Measure cytosolic Ca2+ concentration in several conditions:

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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+

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Thank you