Fluorescent proteins Flashcards
GFP (organism obtained, when, structure, function, spectra, excitation mechanism, application, benefits)
Obtained from Aequorea victoria jellyfish and discovered in the 1960s. In the jellyfish it has a protein aequoporin that absorbs purple light and with a Ca2+ cofactor emit blue light which activates GFP so it fluoresces (the light emitted) green and scares predators
Jablonski diagram: Absorb light at a specific wavelength (blue 475nm and UV 395nm; higher peak at UV in absorption spectra) which excites electron to be ionised at a higher energy level, then almost immediately after drops back down multiple vibrational states to ground state, releasing the remaining energy as light and heat (vibrations of electron).
Since some of the energy the photon had was used to excite electron and heat, the wavelength it emits has less energy and so a longer wavelength, so a different colour emitted (green 509nm)
Structure:
11 beta strands forms beta barrel (shields fluorophore to allow development and maintenance of fluorescence) with diameter 30A, length 40A. Alpha helical caps and irregular alpha helical segment is scaffold for fluorophore in center of cylinder
Applications:
Label proteins (fuse to GFP with linker at N or C terminus) to track their location in cells and tissues with a laser scanning confocal microscope.
Use to study protein dynamics in cell
Benefits:
Requires no cofactors (needs oxygen)
Genetically encoded and can be expressed in most cell types
GFP mutants (mutation, benefits, limitations)
eGFP:
S65T: More blue absorption (one peak)
Five-fold brighter
F64L: Improves efficiency of protein maturation at 37C by 3 fold (before took 2-4 hours to fold)
BFP: Y66H
CFP: Y66W
YFP: T203Y
Mutation far from fluorphore (A206K) prevents dimerisation at high protein concentrations
Additional GFP-like proteins in nature and mutations and benefits
Isolated from non-bioluminescent reef corals
and sea anemones (Anthozoa) for wider colour range.
DsRed was isolated from the sea anemone Discosoma striata
Benefits:
-Allows multicolour imaging
-Autofluorescence is significant reduced in this spectral region, allowing probes to detect deeper into biological tissue
-Living cells/tissues also better tolerate illumination by longer excitation wavelengths (less energy), allowing extended periods for imaging
Mutants:
DsRed tends to form oligomers leading to protein aggregation. Site directed mutagenesis with directed evolution produced monomeric RFP1
Saturation mutagenesis of mRFP1 fluorphore produced a range of FPs emiting 540nm-610nm (mFruits; mCherry, mBanana, ETC)
The most useful mCherry (610 nm emission peak) and mStrawberry (596 nm emission peak), have intrinsic brightness levels of ∼50% and ∼75% that of EGFP, respectively
mCherry is more photostable than mStrawberry, so it is the preferred choice for cell imaging
Considerations when using FPs
-Correct codon usage for improved expression of the FP in the cell/organism of choice (usually made by gene synthesis)
-FP-tagging of the protein of interest – N- or C-terminus?
-EGFP fluorescence shows sensitivity to pH extremes. This has instigated the search for new variants that minimize this drawback
-EGFP fluorescence is oxygen-dependent. Limits its use under anaerobic conditions
-Photostability of FPs can be an issue. EGFP is among the brightest and most photostable of the Aequorea-based FPs. However, the photobleaching properties of FPs can be exploited
Photobleaching: definition, cause and experiments utilising, limitations
Refers to when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage when irradiated extensively using high levels of light/laser power
Largely due to the generation of free oxygen radicals in the cell that attack and permanently destroy the light-emitting properties of the fluorophore
Study protein dynamics:
-Fluorescence Recovery after Photobleaching (FRAP)
-Fluorescence Loss In Photobleaching (FLIP) and inverse FRAP (iFRAP)
Photobleaching techniques, such as FRAP and FLIP require high laser powers and and can be relatively harsh since they require repeated illumination to completely eradicate active fluorophores
What is FRAP, results obtained, what this tells you
Fluorescence Recovery after Photobleaching (FRAP) exploits the ability of laser scanning confocal microscopes to rapidly and irreversibly photobleach a small region of interest (ROI) within the cell
Non-bleached FPs migrate into the ROI from adjacent regions. The recovery of fluorescence in the ROI is then measured as a function of time
The rate of influx of non-bleached proteins provides an estimation of protein mobility and diffusion. Obtain a graph of fluorescence before, during and after photorecovery. Will plateau at fluorescence below intensity pre-bleaching, which indicates the immobile fraction in the region
What is FLIP and iFRAP, results obtained, limitations
Fluorescence Loss In Photobleaching (FLIP) and inverse FRAP (iFRAP) are modifications of the FRAP technique
They measure loss of fluorescence in a ROI after photobleaching of an adjacent region
FLIP: repeated bleaching of one area depletes the
fluorescence of FPs that move through this area.
Fluorescence in the ROI is monitored.
iFRAP: Total fluorescent signal in a cell is bleached except for one area in which the signal
is recorded as a function of time
FLIP and iFRAP approaches acquire measurements from regions of cells that are not being photobleached
Approaches are informative but somewhat limited in tracking localization in specific regions
Optical highlighters: use, classes, mutations
Investigate track localization dynamics
Photoactivateable FPs
PA-GFP: T203H (affects conformation of Gln222 beside the fluorophore and alters the absorbance properties)
Absorbance spectra has one peak at violet (405nm)
which converts the protein into a form (Gln222 decraboxylated) capable of absorbing blue light (spectra has smaller peak at 405nm and large at 484nm). Now, blue light excitation results in green fluorescence
Photoconvertable FPs: irreversibly converts from one fluorescent colour to another
Dendra2 fluoresces green with blue light excitation. Violet light (cleavage at His 62 in His-Tyr-Gly fluorophore) causes it to photoconvert to a red fluorescent form.
Photoswitchable FPs:
Reversibly switch between fluorescent and non-fluorescent forms
Dronpa can switch between a fluorescent cis state (induced by cyan) and a non-fluorescent dark protonated trans state (induced by violet). Can be repeated hundreds of times
Experiments performed with optical highlighters and drawbacks
Photoactivatable FPs:
-Track dynamics in molecular subpopulations:
ROI is irradiated with violet light (5s) to activate. GFP fluorescence is visualised with blue light excitation.
After 5mins, the PA-GFP diffuses out ROI
After 60mins, cytosolic actin, ruffles, and filamentous actin become brighter due to protein movement.
Drawback: non-activated form is not readily distinguishable before photoactivation with 405 nm, making it difficult to identify regions expressing the FP
-Photoactivation localisation microscopy (PALM):
Individual molecules are randomly photoactivated (stochastic activation) and localised with high precision. After their photobleaching a new subset of molecules are activated and their position recorded.
Final image of superimposition of images creates a density map of all fluorophore molecule positions.
Photoswitchable FPs:
-Single molecule super resolution microscopy: Perform repeated cycles of photoactivation (of a individual of FPs) followed by imaging and bleaching to sequentially detect molecules. Merging all images together produces a super-resolution image
Several super-resolution techniques have been developed. They offer the possibility to visualize the distribution, dynamics of individual molecules at a resolution down to ~ 10 nm
Use of fluorescent proteins to track protein dynamics
Bimolecular fluorescence complementation (BiFC):
Venus FP can be split into 2 seperate halves between 7th and 9th beta strands.
N-terminal BiFC fragment (VN) is fused to interacting protein X
C terminal BiFC fragment (VC) is fused to interacting protein Y
When proteins X and Y interact, FP fluorescence is reconstituted
Uses:
-Detects transient interactions and low affinity complexes
Drawbacks:
-High background fluorescence due to self assembly of FP
-FP fragments can be prone to interfering with protein folding and cause can aggregation
-FP assembly is irreversible so not useful for studying interaction dynamics
Instead, smaller fragments have been used to create a TRIPARTITE system that utilises interactions between beta strands 10 and 11 of GFP:
When proteins A and B interact, beta-strand 10 and 11 are tethered. Fluoresence is produced only when acceptor fragment (GFP1-9) is present. This alleviates issues with backgroud fluorescence
Fluorescence Resonance Energy Transfer (FRET):
Resonance energy transfer between FPs can be used to measure the proximity of two molecules in a process known as FRET
One FP molecule (ex. CFP) is called the donor whereas the other (ex. Venus) is called the acceptor
The energy of donor emission MUST be an energy that the acceptor can absorb i.e., the emission spectrum from the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore
No FRET occurs if the donor and acceptor are more than 10 nm apart. Examples of donor and acceptor fluorophores are CFP and Venus, respectively
Reversible so useful for studying interaction dynamics
Chemistry of fluorophore maturation
The tripeptide Ser65-Tyr66-Gly67 is modified post-translationally
Torsional side-chain bond adjustments relocate the side chain of Ser65 in close proximity to the amino nitrogen atom of Gly67
Nucleophilic attack from the Ser65 carbonyl on the amide nitrogen of Gly67 leads to rapid cyclization and formation of an imidazolinone ring system
Loss of water then forms the imidazolin-5-one intermediate
Cyclization is succeeded by a much slower rate-limiting oxygenation of the Tyr66 hydroxybenzyl side chain resulting in the 4-(p-hydroxybenzylidene)-imidazolidin-5-one structure. The double bond that results from this series of reactions results in the linkage of the two π-systems of the rings, forming a larger conjugated system essential for fluorescence
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