Shreya Ray

20091069

11th Nov, 2011

PROTEIN PHOTOACTIVATION

What is Photoactivation?

Photoactivation is any change in the activity of a protein brought about by irradiating it.

In the context of Biophysics, protein photoactivation mostly refers to the change in fluorescent profiles of a protein upon irradiation with light of a certain frequency. This property of certain proteins serve as useful probing tools in Biophysics.

What is Photoactivation?

Fluorescent Proteins

• Fluorescent proteins exhibit fluorescence due to the presence of a fluorophore in the structure, which is responsible for fluorescing.

• One can artificially attach a fluorophore to a protein to make it fluoresce.

• Suppose our fluorophore itself is a protein, then it can be genetically encoded alongside the protein we want it to get attached to, so that every time the protein under study is synthesized, fused to it, the fluorophore will be synthesized too.

Protein as Fluorophore

But are there such proteins?

Green Fluorescent Proteins

Green Fluorescent Proteins (GFPs) were first discovered in the jellyfish Aequorea victoria.

A protein called aequorin interacts with Ca2+ ions, inducing a blue glow.

Some of this luminescent energy is transferred to the GFP which fluoresces green light upon absorbing blue light.

Overall color is shifted towards green.

Fluorophore

Beta-can

A slight change in the structure of Beta-can can give a new fluorescent protein- green, yellow, orange, red

Modified GFPs

Photoactivation

• Kaede protein was discovered to emit red fluorescence after being left on the bench and exposed to sunlight. Kaede, which is originally green fluorescent, after exposure to UV light is photoconverted, becoming red fluorescent.

• EosFP : Gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii was cloned a in Escherichia. It was found to emit strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at ~390 nm due to a photoinduced break in the peptide backbone next to the fluorophore.

• A red-emitting, green-absorbing fluorescent state of GFP can be generated by photoactivation with blue light easily with a laser or fluorescence microscope lamp under conditions of low oxygen concentration.

• These processes are irreversible.

Photoactivation of GFP

Photoactivation of GFP in vivo. GFP-labelled E. coli cells appear as viewed through fluorescein (left) and rhodamine (right) filter sets. GFP in cells on the lower half of the images has been photoactivated by exposure to 475–495 nmlight. Photoactivated cells appear yellow through the fluorescein filters, because they emit continuously from green to red with local maxima at 510, 560, 590 and 600 nm.

Photoactivation of GFP

absorption

emission

Application: Protein Marker

• We can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be “turned on” by photoactivation.

• Suppose we want to track the fate of a particular set of protein molecules as compared to all the other molecules of that protein in the cell, we can genetically tag the proteins with photoactivatable fluorescent proteins. Then, we can photoactivate a small patch using a laser, and then view the progress of the molecules that were present in that patch since now they appear against the background in a different colour.

• This process is mostly used to find protein diffusion rates.

• Protein photoactivation is also generally used to obtain red fluorescence in fluorescence spectroscopy/sorting techniques.

Application: Protein Marker

Application: Protein Marker

(A) Circular region of interest selected with an Olympus FV1000 tornado scanner is illuminated at 405 nm for 5 seconds, t=0 minutes.(B) The photoactivated actin chimera first translocates to the ruffles at the cellular margins as fluorescence intensity decreases in the activated region, t=5 minutes. (C) Ruffles, cytoplasmic actin pools and the filamentous actin network gain more intensity at t=60 Minutes.

(D) Photoconversion of a single mitochondrion (red) in a selected region at 405 nm illumination, t=0 minutes.(E) Close approach of a non-converted (green) mitochondrion (arrow), t=10 minutes. (F) Cargo exchange between mitochondria (arrow), t=20 minutes. (G-I) Examination of lamellipodiawith Dendra2-actin-C-7 in OK cells.

(J) Actin network imaged with a 488-nm laser, t=0 minutes. (K) After completely photoswitching the labeled actin ‘off’ at 488 nm, the region spelling FV10 was activated with a 405 nm laser, t=3 minutes.(L) FV10 region photobleached while imaging the actin network at 488 nm.

Application: Two-colour super-resolution microscopy using PALM

Stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached.

Thus, very closely spaced molecules that reside in the same diffraction-limited volume (and would otherwise be spatially indistinguishable are temporally separated.

We merge all of the single-molecule positions obtained by repeated cycles of photoactivation, follow by imaging and bleaching to produce the final super-resolution.

Application: Two-colour super-resolution microscopy using PALM

The prior knowledge that the diffraction-limited image of a molecule originates from a single source enables the estimation of the location (center) of that molecule with a precision well beyond that of the diffraction limit.

The molecular coordinates are not actually represented by a single point to identify their spatial position, rather a Gaussian intensity distribution corresponding to the positional uncertainty of their location is employed to build the image map.

A cooled electron-multiplying charge-coupled device (EMCCD) camera system.

Central to the instrumentation is a high-speed computer system that acquires and processes images recorded by the camera.

Application: Two-colour super-resolution microscopy using PALM

There is very little direct overlap between actin and paxillin (white arrowheads), although actin bundles densely cluster around several fibrillar paxillin adhesions (white arrows). In widefield TIRF imaging, actin and paxillin appear to co-localize in focal adhesions, contrary to the information afforded by super-resolution microscopy.

Application: Two-colour super-resolution microscopy using PALM

• To overcome the diffraction barrier we may employ photo-switchable fluorescent probes to resolve spatial differences in dense populations of molecules with super-resolution.

• Pulse-Chase experiments with super-resolution

• Live-Cell Imaging with PALM

- very fast switching

- more intense light

- disadvantages of photobleaching

Application: In Two-photon microscopy

• If paGFP-labeled signaling molecules are visualized by means of two-photon activation, the position of the activation volume in the tissue can be determined precisely.

• An organelle can be marked via photoactivation.

• Two-photon microscopy allows us to image layers at various depths.

• All the local information can be combined to get the full 3D picture of the organ.

Application: light-based mapping of motor cortex

• Traditionally, mapping the motor cortex requires electrodes to stimulate the brain and define motor output pathways. Although effective, electrode-based methods are labour-intensive, potentially damaging to the cortex and can have off-target effects. As an alternative method of motor mapping, we photostimulated transgenic mice expressing the light-sensitive ion channel channelrhodopsin-2 in predominantly layer-5 output cortical neurons. We report that optical stimulation of these neurons in vivo using a stage scanning laser system resulted in muscle excitation within 10-20 ms, which can be recorded using implanted electromyogram electrodes or by a non-invasive motion sensor. This approach allowed us to make highly reproducible automated maps of the mouse forelimb and hind limb motor cortex much faster than with previous methods. We anticipate that the approach will facilitate the study of changes in the location and properties of motor maps after skilled training or damage to the nervous system.

Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.

Ayling OG, Harrison TC, Boyd JD, Goroshkov A, Murphy TH.

Application: A Photoactivation ‘System’

Elizabeth Pham, Evan Mills and Kevin Truong

• Through tightly regulated Ca2+ concentration fluxes varying in location, time, frequency, and amplitude, Ca2+ signals regulate diverse and distinct physiological processes.

• The ability to generate local and global Ca2+ signals would allow us to better study the Ca2+ specificity problem and to engineer synthetic proteins that respond to specific Ca2+ signals.

• Using light to generate these Ca2+ signals is particularly desirable because light causes minimal damage to live cells and has the potential for high temporal and spatial resolution.

• Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to reversibly generate local or global Ca2+ signals.

Application: A Photoactivation ‘System’

With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca2+ channel, within seconds, generating a local Ca2+ signal on the plasma membrane, and returning to the cytoplasm after 10sec.

A structural change produced by exposure to blue light brings about a change in the binding ability.

Application: A Photoactivation ‘System’

We begin with an already photoactivated protein and study its interaction with the receptors under various conditions. Whenever the protein binds receptors, the cell membrane becomes wholly red, since the colour red dominates over the greenish colour of the fluorescent tag of the membrane.

Reversible calcium flux (false coloured)…

Application: Photolithography

Purified EosFP was cosslinkedby paraformaldehyd on a nitrocellulosemembrane. Based on a photography ofAlbert Einstein (upper left), a mask wascreated. The upper right picture showsthe unconverted green fluorescentprotein on the membrane under themask. After irradiation for 3 min with UVlight, the membrane was photographed(lower left). The red fluorescence of thephotoconverted protein in the lower rightpicture was excited at 540 nm andphotographed using a long pass filterwith a cut off at 590 nm.

**An interesting prospective for data storage on the molecular level

• Wiedenmann et al.- From EosFP to mIrisFP: structure-based development of advanced photoactivatable marker proteins of the GFP-family.

• George H. Patterson -Photoactivation and Imaging of Photoactivatable Fluorescent Proteins

• Advances in fluorescent protein technologyNathan C.

• Elizabeth Pham, Evan Mills and Kevin Truong- A Synthetic Photoactivated Proteinto Generate Local or Global Ca2+ Signals

• John Runions, Thorsten Brach, Sebastian Ku¨hner and Chris Hawes-Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane.

• Fuchs, Wiedenmann, Nienhaus et al. A photoactivatable marker protein for pulse-chase imaging with super-resolution

• www.scholarpedia.org

• www.wikipedia.org

References