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Fluorescent Protein-Based Tools for Neuroscience

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Fluorescent Protein-Based Tools for Neuroscience !1 !2 Fluorescent protein-based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent proteins (FPs) Robert E. Campbell Department of Chemistry Other fluorophore technologies Single FP-based biosensors Imaging Structure & Function in the Nervous System Cold Spring Harbor, July 31, 2019. Lots of structural Lots of structural information information & Transmitted light Fluorescence microscopy of fluorescent color microscopy of live cells live cells No molecular provides molecular information information (more colors = more information) !5 !6 Fluorescence microscopy requires fluorophores Non-natural fluorophores for protein labelling Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N 495 nm 519 nm 557 nm 576 nm - - CO2 CO2 S O O O N O N C N N C S ϕ = quantum yield - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N Fluorescein Tetramethylrhodamine ε = 73,000 M-1cm-1 C S (FITC) (TRITC) A non-natural fluorophore must be chemically linked to Non-natural fluorophores made by chemical synthesis a protein of interest… !7 !8 Getting non-natural fluorophores into a cell Some sea creatures make natural fluorophores Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N Bioluminescent - Fluorescent CO2 CO -- S 2 S C N NHN C H S S Chemically labeled proteins of interest Microinjection with micropipet O O O N O N Fluorescent - CO2 CO - S 2 N NH H S …and then manually injected into a cell Some natural fluorophores are genetically encoded proteins http://www.luminescentlabs.org/and can be transplanted into cells as DNA! 228 OSAMU SHIMOMURA, FRANK H. JOHNSON AND YO SAIGA respect from the greenish luminescence of ing effect in the luminescent recation by the whole organism or fresh squeezates.a adding 5 ml of 0.01 M salt solution to The light-emitting potency was calculated 0.05 ml of aequorin in 0.01 M EDTA-2Na to be 42,500 L.U./mg, which is equivalent solution at pH 6.0. Among them, no acti- to the total light of 6.6 pg of Cypridina vation was found with ions of magnesium, luciferin whose molecular weight is 469 barium, potassium, ammonium, zinc, co- (Hirata, Shimomura and Eguchi, '59). balt, manganese, ferric or ferrous iron, Ultraviolet absorption spectrum copper, or lead. A slight activation that Except for a slight bulge at 310 mw, the occurred with cadmium was probably due ultraviolet absorption spectrum (fig. 3) is to the presence of impurities, inasmuch as similar to that of simple proteins, with a the purest available substance had the peak at 280 mp. After the luminescent re- least effect. Some activating effect of stron- action the bulge at 310 mw disappears and tium could either be real or due to impuri- a new absorption maximum shows up at ties; the evidence is not conclusive. !9 333 mu. In regard to calcium, the influence of !10 concentration is illustrated by the data Requirement of Cu++ for lumines- shown in figure 4,(A). When the concen- The discovery of a protein fluorophore The discovery of a protein fluorophorecence, and effects of other cations 3 A protein giving solutions that look slightly green- ish in sunlight though only yellowish under tungsten Extraction, Purification and Properties of Aequorin, The calciumExtraction, Purification requirement and Properties has of Aequorin, been re- lights, and exhibiting a very bright,. greenish fluores- a Bioluminescent Protein from the Luminous ferred toa Bioluminescent above. Thirteen Protein from the other Luminous cations in cence in the ultraviolet of a Mmerapte, has also been Hydromedusan, Aequorea’ isolated from squeezates. No mdicatlons of a lummes. Hydromedusan, Aequorea’ the form of salts of chloride, sulfate or cent reaction of this sustance could be detected. OSAMU SHIMOMUFL4,2 FUNK H. JOHNSON AND YO SAIGA Department of Biology, Princeton University, Princeton, New Jersey, Studies of the ern!ssion spectra of both this protem acetate were testedand the Fridayfor Harbor a Laboratories, possible University of Washington, activat- OSAMU SHIMOMUFL4,2 FUNK H. JOHNSON AND YO SAIGA Friday Harbor, Washington and aequorin are m progress. Department of Biology, Princeton University, Princeton, New Jersey, of and the Friday Harbor Laboratories, University Washington, noctiluca, and observed that luminous Friday Harbor, Washington In experiments that have become classic 228in bioluminescence, OSAMU Dubois SHIMOMURA, (1885, FRANK1887), H.slime JOHNSON from AND the YObell SAIGA could be rubbed onto first prepared from a luminous elaterid, various surfaces making them glow as if respectPyrophorus, from theand greenish a luminous luminescence clam, Pholas, of ingon effect fire. inSpallanzani the luminescent (1794, recation 1798) notedby therespectively, whole organism crude orextracts fresh squeezates.acontaining a addingthat luminescence5 ml of 0.01 ofM thissalt organismsolution tocon- Thesubstrate, light-emitting luciferin, potency and anwas enzyme, calculated luci- 0.05tinued ml ofafter aequorin death, in and 0.01 that M aEDTA-2Na dark, almost noctiluca, and observed that luminous liquefied specimen luminesced on addition In experiments that have become classic toferase, be 42,500 which L.U./mg, luminesced which onis equivalent mixing in solution at pH 6.0. Among them, no acti- 228in bioluminescence, OSAMU Dubois SHIMOMURA, (1885, FRANK1887), H.slime JOHNSON from AND the YObell SAIGA could be rubbed onto toaqueous the total solution light containingof 6.6 pg ofdissolved Cypridina oxy- vationof fresh was foundwater. withAt ionsthe turnof magnesium, of the nine- various surfaces making them glow as if luciferingen. In whosethe years molecular that have weight followed, is 469 ef- barium,teenth potassium,century, vonammonium, Humboldt zinc, (1799-co- first prepared from a luminous elaterid, (Hirata,forts have Shimomura repeatedly and been Eguchi, made '59). to sepa- balt,1804; manganese, cf. von ferricHumboldt, or ferrous 1853) iron, and on fire. Spallanzani (1794, 1798) noted rate functionallyUltraviolet absorption similar components spectrum from copper,Macartney or lead. (1810) A slight found activation that lumines- that respectPyrophorus, from theand greenish a luminous luminescence clam, Pholas, of ing effect in the luminescent recation by numerousExcept for other, a slight divers bulge types at 310 of mw,luminous the occurredcence ofwith medusae cadmium could was be probably elicited bydue elec- ultraviolet absorption spectrum (fig. is that luminescence of this organism con- organisms (Harvey, ’52; ’55). Although3) to tricalthe presence stimuli, of a impurities,phenomenon inasmuch that has as only therespectively, whole organism crude orextracts fresh squeezates.acontaining a adding 5 ml of 0.01 M salt solution to similar to that of simple proteins, with a therecently purest beenavailable investigated substance from had a modernthe Shimomura isolated and, tinued after death, and that a dark, almost peakthe majorityat 280 mp. of Afterthese the efforts luminescent proved re- un- least effect. Some activating effect of stron- Thesubstrate, light-emitting luciferin, potency and anwas enzyme, calculated luci- 0.05 ml of aequorin in 0.01 M EDTA-2Na successful, about a dozen biologically spe- viewpoint (Davenport and Nicol, ’55; liquefied specimen luminesced on addition action the bulge at 310 mw disappears and tiumNicol, could ’60). either Macartney be real or (1810) due to impuri-also found to be 42,500 L.U./mg, which is equivalent solution at pH 6.0. Among them, no acti- acific, new chemicallyabsorption differentmaximum luciferin-lucif shows up at er- ties; the evidence is not conclusive. ferase, which luminesced on mixing in ase systems have by now been obtained in that no diminution in luminescence could of fresh water. At the turn of the nine- 333 mu. beIn detectedregard to in calcium, a vacuum, the asinfluence compared of to to the total light of 6.6 pg of Cypridina vation was found with ions of magnesium, varying degrees of purification (Johnson, over decades, performed aqueous solution containing dissolved oxy- concentration is illustrated by the data teenth century, von Humboldt (1799- Sie Requirementand Haneda, of’61). Cu++ The for presentlumines- inves- aerobic conditions. The lack of a free luciferin whose molecular weight is 469 barium, potassium, ammonium, zinc, co- shownoxygen in figurerequirement 4,(A). forWhen luminescence the concen- was gen. In the years that have followed, ef- tigationcence, has resulted and effects in the of otherdiscovery of a convincingly demonstrated by Harvey (‘26; (Hirata, Shimomura and Eguchi, '59). balt,1804; manganese, cf. von ferricHumboldt, or ferrous 1853) iron, and new type of luminescentcations system, differing forts have repeatedly been made to sepa- 3Harvey A protein andgiving Korr, solutions ’38), that notlook slightlyonly for green- med- Ultraviolet absorption spectrum Macartney (1810) found that lumines- from those hitherto extracted in being ish in sunlight though only yellowish under tungsten copper, or lead. A slight activation that The calcium requirement has been re- lights,usae and but exhibiting also radiolarians a very bright,. and greenish ctenophores. fluores- extensive studies on this rate functionally similar components from ferredcomprised to above. of a Thirteensingle organic other cationscomponent, in cence in the ultraviolet of a Mmerapte, has also been Except for a slight bulge at 310 mw, the cence of medusae could be elicited by elec- isolatedLuminescence from squeezates. under No mdicatlons strictly of a lummes.anaerobic occurred
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