1 2 Fluorescent -based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent (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 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 = - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N 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, absorption spectrum copper, or lead. A slight activation that Except for a slight bulge at 310 mw, the occurred with 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- , 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 aLaboratories, 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 or extracts fresh squeezates.a containing a addingthat luminescence5 ml of 0.01 ofM thissalt organismsolution tocon- Thesubstrate, light-emitting luciferin, potency and anwas , 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 ferric Humboldt, 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 or extracts fresh squeezates.a containing 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 modern the 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 chemically absorption 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 ferric Humboldt, or ferrous 1853) iron, and new type of luminescentcations system, differing forts have repeatedly been made to sepa- 3Harvey A protein and giving 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 Thirteen single organic other cations component, 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 with cadmium was probably due thewith form the propertiesof salts of ofchloride, a protein. sulfate In aque-or cent reaction of this sustance could be detected. numerous other, divers types of luminous Studiesconditions, of the ern!ssion however, spectra is ofan both unusual this protem feature ultraviolet absorption spectrum (fig. 3) is to tricalthe presence stimuli, of a impurities,phenomenon inasmuch that has as only acetateous solution were testedeither fordevoid a possible of, or saturatedactivat- andamong aequorin the are mvarious progress. types of luminescent organisms (Harvey, ’52; ’55). Although with, oxygen, this protein gives a light- similar to that of simple proteins, with a therecently purest beenavailable investigated substance from had a modern the systems now known (Harvey, ’52; ’55). the majority of these efforts proved un- emitting reaction on addition of Ca++. Though unsuccessful in attempts to ob- peak at 280 mp. After the luminescent re- leastviewpoint effect. Some (Davenport activating andeffect Nicol,of stron- ’55; With the procedures employed, nearly tain a luciferin - reaction with Green Fluorescent successful, about a dozen biologically spe- 10,000 individual specimens of the hydro- action the bulge at 310 mw disappears and tiumNicol, could ’60). either Macartney be real or (1810) due to impuri-also found extracts of Aequorea, Harvey (’21) found cific, chemically different luciferin-lucif er- medusan, Aequorea, yielded about 5 mg of that the photogenic tissues dried over a new absorption maximum shows up at ties;that the no evidence diminution is not in conclusive. luminescence could the highly purified, active substance which CaCL would luminesce when moistened. ase systems have by now been obtained in we have named “Aequorin” (Shimomura, Moreover, strips of the margin of the um- 333 mu. beIn detectedregard to in calcium, a vacuum, the asinfluence compared of to Johnson and Saiga, ’62). varying degrees of purification (Johnson, brella, where the photogenic organs are concentrationaerobic conditions. is illustrated The bylack the of dataa free While certain aspects of the phenome- ~ Protein (GFP) Sie Requirementand Haneda, of’61). Cu++ The for presentlumines- inves- non of luminescence in medusae have Received Jan. 19, ’62. Accepted Feb. 8. ’62. shown in figure 4,(A). When the concen- 1 Aided in part by National Science Foundation oxygen requirement for luminescence was long been known, the in- grant number 6-12857 Office of Naval Research Con- tigationcence, has resulted and effects in the of otherdiscovery of a volved has remained very much of an tract Nonr 1353(00),’Project NR 165-253 and the Eugene Higgins Fund allocated to Princetdn Univer- convincingly demonstrated by Harvey (‘26; enigma. As early as the first century, sity. Publication in whole or in part by or for any new type of luminescentcations system, differing 3 A protein giving solutions that look slightly green- purpose of the United States Government is permitted. Harvey and Korr, ’38), not only for med- Pliny (cf. Harvey, ’57) described the light 2On leave from the Pharmacy Department, Uni- from those hitherto extracted in being ish in sunlight though only yellowish under tungsten of “Pulmo Marinus,” evidently Pelagia versity of Nagasaki, Japan. The calcium requirement has been re- lights,usae and but exhibiting also radiolarians a very bright,. and greenish ctenophores. fluores- ferredcomprised to above. of a Thirteen single organic other cations component, in cence in the ultraviolet of a Mmerapte, has also been 223 In aque- isolatedLuminescence from squeezates. under No mdicatlons strictly of a lummes. anaerobic thewith form the propertiesof salts of ofchloride, a protein. sulfate or centconditions, reaction of however,this sustance is ancould unusual be detected. feature ous solution either devoid of, or saturated Studies of the ern!ssion spectra of both this protem 228 OSAMU SHIMOMURA,acetate FRANK were tested H. for JOHNSON a possible activat- AND andamong aequorinYO theSAIGA are mvarious progress. types of luminescent with, oxygen, this protein gives a light- systems now known (Harvey, ’52; ’55). emitting reaction on addition of Ca++. Though unsuccessful in attempts to ob- With the procedures employed, nearly tain a luciferin - luciferase reaction with 10,000 individual specimens of the Shimomurahydro- et al., J. Cell. Comp. Physiol. , 59, 223. Shimomura et al., J. Cell. Comp. Physiol. , 59, 223. respect from the greenish luminescence of extracts of Aequorea, Harvey (’21) found 1962 Ultraviolet absorption spectra of aequorin the product of the luminescent 1962 ing effect in the luminescent recation by Fig. 3 (A), medusan, Aequorea, yielded about 5 mg of reaction (B), and the product of the reaction between aequorin and NaHS03 (C). The con- that the photogenic tissues dried over centration of aequorin used for curve A was 0.41 mg/ml by dry weight; for curve B, 20 pl the whole organism or fresh squeezates.athe highly purified, adding active substance 5 ml which of 0.01 M salt solution to of 8% Ca(CH3C00)2Hz0solution were added to a 3 ml portion of aequorin solution to CaCL would luminesce when moistened. complete the luminescent reaction; for Curve C, 10 pl of 10% NaHSOs solution were added The light-emitting potency was calculatedwe have named “Aequorin”0.05 ml (Shimomura, of aequorin Moreover, in 0.01strips of Mthe EDTA-2Namargin of the um- to a 1 ml portion of aequorin solution. Johnson and Saiga, ’62). brella, where the photogenic organs are to be 42,500 L.U./mg, which is equivalentWhile certain solutionaspects of the at phenome- pH 6.0. Among~ them, no acti- non of luminescence in medusae have Received Jan. 19, ’62. Accepted Feb. 8. ’62. to the total light of 6.6 pg of Cypridina 1 Aided in part by National Science Foundation long been known,vation the biochemistry was found in- grantwith number ions 6-12857 of Office magnesium, of Naval Research Con- volved has remained very much of an tract Nonr 1353(00),’Project NR 165-253 and the luciferin whose molecular weight is 469 barium, potassium,Eugene ammonium, Higgins Fund allocated tozinc, Princetdn co- Univer- enigma. As early as the first century, sity. Publication in whole or in part by or for any purpose of the United States Government is permitted. (Hirata, Shimomura and Eguchi, '59).Pliny (cf. Harvey,balt, ’57) describedmanganese, the light 2On ferric leave from or the ferrous Pharmacy Department, iron, Uni- of “Pulmo Marinus,” evidently Pelagia versity of Nagasaki, Japan. Fig. 3 Ultraviolet absorption spectra of aequorin (A), the product of the luminescent Ultraviolet absorption spectrum copper, or lead. A slight activation that reaction (B), and the product of the reaction between aequorin and NaHS03 (C). The con- Except for a slight bulge at 310 mw, the occurred with cadmium was probably due223 centration of aequorin used for curve A was 0.41 mg/ml by dry weight; for curve B, 20 pl ultraviolet absorption spectrum (fig. 3) is to the presence of impurities, inasmuch as of 8% Ca(CH3C00)2Hz0solution were added to a 3 ml portion of aequorin solution to similar to that of simple proteins, with a the purest available substance had the complete the luminescent reaction; for Curve C, 10 pl of 10% NaHSOs solution were added peak at 280 mp. After the luminescent re- least effect. Some activating effect of stron- to a 1 ml portion of aequorin solution. 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. 333 mu. Fig. 3 Ultraviolet Inabsorption regard spectra ofto aequorin calcium, (A), the product the of influencethe luminescent of 11 12 reaction (B), and the product of the reaction between aequorin and NaHS03 (C). The con- centration of aequorinconcentration used for curve A was 0.41is mg/mlillustrated by dry weight; forby curve the B, 20 pldata Requirement of Cu++ for lumines-of 8% Ca(CH3C00)2Hz0solution were added to a 3 ml portion of aequorin solution to complete the luminescentshown reaction; in forfigure Curve C, 104,(A). pl of 10% NaHSOsWhen solution the were concen- added 30 years later, the gene for GFP was cloned by Prashercence, and effects of other to a 1 ml portion of aequorin solution. Aequorea jellyfish green fluorescent protein

cations 3 A protein giving solutions that look slightly green- ish in sunlight though only yellowish under tungsten The calciumExtraction, Purification requirement and Properties has of Aequorin, been re- lights, and exhibiting a very bright,. greenish fluores- 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. 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 aLaboratories, possible University of Washington, activat- Friday Harbor, Washington and aequorin are m progress.

In experiments that have become classic noctiluca, and observed that luminous 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 Gone, 111 (1992) 229-233 respectPyrophorus, from theand greenish a luminous luminescence clam, Pholas, of ingon effect fire. inSpallanzani the luminescent (1794, recation 1798) notedby © 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.001962 - 1992 229 1928 - 1952 - therespectively, whole organism crude or extracts fresh squeezates.a containing 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 GENE 06296 2018 2016 liquefied specimen luminesced on addition toferase, be 42,500 which L.U./mg, luminesced which onis equivalent mixing in solution at pH 6.0. Among them, no acti- toaqueous the total solution light containingof 6.6 pg ofdissolved Cypridina oxy- vationof fresh was foundwater. withAt ionsthe turnof magnesium, of the nine- luciferin whose molecular weight is 469 barium,teenth potassium, century, vonammonium, Humboldt zinc, (1799-co- gen. In the years that have followed, ef- Primary structure of the Aequorea victoria green-fluorescent protein (Hirata,forts have Shimomura repeatedly and been Eguchi, made '59). to sepa- balt,1804; manganese, cf. von ferric Humboldt, or ferrous 1853) iron, and rate functionallyUltraviolet absorption similar components spectrum from copper,Macartney or lead. (1810) A slight found activation that lumines- that 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- (Bioluminescence; Cnidaria; aequorin; energy transfer; ; cloning) ultraviolet absorption spectrum (fig. is organisms (Harvey, ’52; ’55). Although3) to tricalthe presence stimuli, of a impurities,phenomenon inasmuch that has as only similar to that of simple proteins, with a therecently purest beenavailable investigated substance from had a modern the peakthe majorityat 280 mp. of Afterthese the efforts luminescent proved re- un- least effect. Some activating effect of stron- successful, about a dozen biologically spe- viewpoint (Davenport and Nicol, ’55; action the bulge at 310 mw disappears and tiumNicol, could ’60). either Macartney be real or (1810) due to impuri-also found Douglas C. Prasher a, Virginia K. Eckenrode b, William W. Ward c, Frank G. Prendergast d and Milton J. Cormier b acific, new chemically absorption differentmaximum luciferin-lucif shows up at er- ties; the evidence is not conclusive. ase systems have by now been obtained in that no diminution in luminescence could 333 mu. beIn detectedregard to in calcium, a vacuum, the asinfluence compared of to Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (U.S.A.); b Biochemistry Department, University of varying degrees of purification (Johnson, concentration is illustrated by the data Georgia, Athens, GA 30602 (U.S.A.) Tel. (404)542-I747; c Department of Biochemistry and Microbiology. Cook College. Rutgers University. Sie Requirementand Haneda, of’61). Cu++ The for presentlumines- inves- aerobic conditions. The lack of a free shownoxygen in figurerequirement 4,(A). forWhen luminescence the concen- was New Brunswick, NJ 08903 (U.S.A.) Tel. (908)932-9562; and u Department of Biochemistry and Molecular Biology. Mayo Foundation, Roch- tigationcence, has resulted and effects in the of otherdiscovery of a convincingly demonstrated by Harvey (‘26; new type of luminescentcations system, differing ester. MN 55905 (U.S.A.) Tel. (507)284-2065 3Harvey A protein and giving Korr, solutions ’38), that notlook slightlyonly for green- med- from those hitherto extracted in being ish in sunlight though only yellowish under tungsten The calcium requirement has been re- lights,usae and but exhibiting also radiolarians a very bright,. and greenish ctenophores. fluores- Received by S.R. Kushner: 21 March 1991 ferredcomprised to above. of a Thirteen single organic other cations component, in cence in the ultraviolet of a Mmerapte, has also been In aque- isolatedLuminescence from squeezates. under No mdicatlons strictly of a lummes. anaerobic Revised/Accepted: 13 September/27 September 1991 thewith form the propertiesof salts of ofchloride, a protein. sulfate or centconditions, reaction of however,this sustance is ancould unusual be detected. feature Received at publishers: 26 November 1991 ous solution either devoid of, or saturated Studies of the ern!ssion spectra of both this protem acetate were tested for a possible activat- andamong aequorin the are mvarious progress. types of luminescent with, oxygen, this protein gives a light- systems now known (Harvey, ’52; ’55). emitting reaction on addition of Ca++. Though unsuccessful in attempts to ob- With the procedures employed, nearly tain a luciferin - luciferase reaction with 10,000 individual specimens of the hydro- extracts of Aequorea, Harvey (’21) found medusan, Aequorea, yielded about 5 mg of that the photogenic tissues dried over SUMMARY the highly purified, active substance which CaCL would luminesce when moistened. we have named “Aequorin” (Shimomura, Moreover, strips of the margin of the um- Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-Wansfer accepters in bioluminescence. GFPs flu- O cyclization Johnson and Saiga, ’62). brella, where the photogenic organs are oresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca2 + -activated pho- Tyr66 Gly67 O O

While certain aspects of the phenome- ~ toprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromopho~'e, which is comprised non of luminescence in medusae have Received Jan. 19, ’62. Accepted Feb. 8. ’62. oxidation 1 Aided in part by National Science Foundation of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA long been known, the biochemistry in- grant number 6-12857 Office of Naval Research Con- N volved has remained very much of an tract Nonr 1353(00),’Project NR 165-253 and the and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfplO cDNA encodes a 238-aa-residue polypeptide Eugene Higgins Fund allocated to Princetdn Univer- H N N enigma. As early as the first century, sity. Publication in whole or in part by or for any with a calculated Mr of 26 888. Comparison of A. victoria GFP genornic clones shows three different restriction enzyme HN O dehydration Pliny (cf. Harvey, ’57) described the light purpose of the United States Government is permitted. O N N 2On leave from the Pharmacy Department, Uni- patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, HO of “Pulmo Marinus,” evidently Pelagia versity of Nagasaki, Japan. HO O O O Washington. The gfp gene encoded by the AGFP2 genomic clone is comprised of at least three exerts spread over 2.6 kb. 223 The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this HO HO HO unique class of proteins. N N N H H H Ser65 mature chromophore

INTRODUCTION phyla have been characterized. Light from luminescent cni- Shimomura, FEBS Lett. 1979, 104, 220; Ormö et al., Science 1996, 273, 1392; Tsien, Annu. Rev. Biochem. 1998, 67, 509; Shimomura et al., J. Cell. Comp. Physiol. , 59,daria 223; is primarily Prashergreen whereas light emitted et fromal., cteno- Gene , 111, 229. Fig. 3 Ultraviolet absorption spectra of aequorin (A), the product of the luminescent Luminescence is common in a1962 variety of marine inver- phores is blue. The green light of cnidaria is due to the 1992 http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ reaction (B), and the product of the reaction between aequorin and NaHS03 (C). The con- presence of a class of proteins called green-fluorescent pro- centration of aequorin used for curve A was 0.41 mg/ml by dry weight; for curve B, 20 pl tebrates. Many cnidarians and probably all ctenophores of 8% Ca(CH3C00)2Hz0solution were added to a 3 ml portion of aequorin solution to emit light when mechanically disturbed. Proteins respon- reins (GFPs). They are highly fluorescent and are activated complete the luminescent reaction; for Curve C, 10 pl of 10% NaHSOs solution were added to a 1 ml portion of aequorin solution. sible for bioluminescence from several species of these two in vivo by an energy transfer process via a luciferase or a Ca2+-activated photoprotein, both of which produce en- ergy during the oxidation of coelenterate-type luciferin. In the cnidarian Aequorea, the photoprotein aequorin excites Correspondenceto: Dr. D.C. Prasher, Redfield Bldg., Woods Hole Oceano- graphic Institution, Woods Hole, MA 02543 (U.S.A.) the GFP by an unknown mechar6sm to release green light. Tel. (508)457-2000, ext. 23 i I; Fax (508)457-2195. Previous studies suggesting that Aequorea GFP is stimu- lated via a radiationless mechanism (Mofise etal., 1974) Abbr~.wiations:A., Aequorea; aa, amino acid(s); bp, base pair(s); GFP, have been questioned (Ward, 1979). The GFP from Re- grecn-fluorescent protein; gfp, DNA or RNA encoding GFP; kb, kilo- nilla, another cnidarian, on the other hand, clearly receives base(s) or 1000bp; nt, nucleofide(s), oligo, oligodenxyribonucleofide; ORFo open reading frame(s). energy from the Renilla luciferase-oxyluciferin excited state Fig. 3 Ultraviolet absorption spectra of aequorin (A), the product of the luminescent reaction (B), and the product of the reaction between aequorin and NaHS03 (C). The con- centration of aequorin used for curve A was 0.41 mg/ml by dry weight; for curve B, 20 pl of 8% Ca(CH3C00)2Hz0solution were added to a 3 ml portion of aequorin solution to complete the luminescent reaction; for Curve C, 10 pl of 10% NaHSOs solution were added to a 1 ml portion of aequorin solution. 13 14 Typical live cell imaging using GFP Natural FP color variants in coral and anemone

fluorescent gene of protein gene interest

mammalian cells with GFP-labeled end-binding protein 3 (EB3)

cyan FP from yellow FP from orange FP from red FP from Clavularia sp. Zoanthus sp. Fungia concinna Discosoma sp.

Matz et al., Nat. Biotechnol. 1999, 17, 969; Henderson & Remington, Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 12712; Remington et al., Biochemistry 2005, 44, 202; Kikuchi et al., Biochemistry 2008, 47, 11573;Yarbrough et al., Proc. Movie and image from Michael W. Davidson; Microtubule schematic from Nakamura et al., PLoS ONE, 2012, 7(12), e51442 Natl. Acad. Sci. U. S. A. 2001, 98, 462. Images: reefguide.org; goldenmarindo.com; www.seascapestudio.net.

15 16 RFP and GFP have similar The trouble with tetramers

DNA Aequorea GFP

correct

O O trafficking

N N N N HO -O O H N 2 N O O DNA Discosoma RFP actin Aequorea GFP Discosoma RFP improper trafficking Shimomura, FEBS Lett. 1979, 104, 220; Gross et al., Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 11990. 17 18 Nature has generously provided us with a Engineering fluorescent proteins variety of genetically encoded fluorophores fluorescent protein gene design and rational e.g. monomerizing mutations; some Rational However, ideal for jellyfish and coral ≠ ideal for research, modification colour-changing mutations engineering so we must engineer fluorescent proteins to suit our needs! Directed repeat with winners evolution error-prone PCR The 'ideal' FP for live cell imaging would… amplification One half of The Nobel …be brightly fluorescent (EC * QY), Prize in Chemistry 2018 was one half awarded …be a monomer, to Frances H. Arnold “for the directed …be red or near- fluorescent, evolution of .” isolate plasmid, …be resistant to photobleaching, gene library express gene, characterize …be insensitive to changes in pH, insert in plasmid & transform E.coli protein …be tolerant of fusion to other proteins, pick it …have fast and efficient folding and maturation, find? the ? …have narrow absorption and emission profiles, ?improved one …have a single exponential lifetime.

19 20 Early examples of engineered GFP variants A palette of monomeric fluorescent proteins

Change this O protein engineering N O residue to other H HN O HO O wtGFPN aromatic amino N HO HO N acids H dimer monomer brighter O

N N O O HO protein N H DsRed DsRed-derived engineering GFP-derived series (mFruit) series

O O O BFP CFPO EGFP YFPOH HN N N N N N N N N N HN O O Nathan Shaner and Paul Steinbach Aequorea GFP

Heim et al., Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 12501; Heim et al., Nature 1995, 373, 663; Campbell et al., Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 7877; Wang et al., Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 16745; Cubitt et al., Trends Biochem. Sci. 1995, 20, 448; Heim & Tsien, Curr. Biol. 1996, 6, 178; Ormö et al., Science 1996, 273, 1392. Shaner et al., Nat. Biotechnol. 2004, 22, 1567; Shu et al., Biochemistry 2006, 45, 9639; Shaner et al., Nat. Methods 2008, 5, 545. C C

Infrastructure: Protein Synthesis: building DNA: storing and reading - 36 B Y

65 -

supporting and new molecular machines genetic information 3 . Molecular Machinery: 0

30 63 moving cells 67. Transfer RNA 4tna 84. DNA 1bna D a

68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease 84 v rcsb.org i 31 37 63. Actin 1m8q d A Tour of the Protein Data Bank EcoRI 1eri 69. Threonyl-tRNA Synthetase 1qf6 S .

64. Myosin 1m8q 70. Glutaminyl-tRNA Synthetase1euq 86. DNA Photolyase 1tez G 65. Microtubule1tub o Cells build many complex molecular machines that perform the biological jobs needed for life. Some of these machines Energy Production: 71. Isoleucyl-tRNA Synthetase 1ffy 87. Topoisomerase 1a36 o d

66. Collagen 1bkv s are molecular scissors that cut food into digestible pieces. Others then use these pieces to build new molecules when 72. Phenylalanyl-tRNA Synthetase 1eiy 88. RNA Polymerase 2e2i e

powering the processes l

(far left) l

73. Aspartyl-tRNA Synthetase 1asy 89. lac Repressor 1lbh 1efa 85 a

cells grow or tissues need to be repaired. Some molecular machines form sturdy beams that support cells, and others are of the cell n 32 74. Ribosome 1j5e, 1jj2 90. Cataabolite Gene Activator d

t motors that use energy to crawl along these beams. Some recognize attackers and mobilize defenses against infection. 30. Cytochrome c Oxidase 75. Elongation Factor Tu/tRNA 1ttt Protein 1cgp h e

(Complex IV) 1oco 76. Elongation Factor G 1dar 91. TATA-binding Protein/ R 31. Cytochrome c 3cyt Transcription Factor IIB 1ais C Researchers around the world are studying these molecules at the atomic level. These 3D structures are 77. Elongation Factor Ts and Tu 1efu S Adenosine 86 B

32. Cytochrome bc1 92. DNA Helicase 4esv freely available at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. A few 78. Prefoldin 1fxk 67 P

Triphosphate D (Complex III) 1bgy 93. DNA Polymerase 1tau (ATP) examples from the ~100,000 structures held in the PDB are shown here at a magnification of about 33. Succinate Dehydrogenase 79. Chaperonin GroEL/ES 1aon B 80. Proline cis/trans Isomerase 2cpl 94. Nucleosome 1aoi 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is (Complex II) 1nek 95. HU Protein 1p51 Glucose 34. NADH-Quinone Oxidoreductase 81. Heat Shock Protein Hsp90 2cg9 illustrated here, from the water molecule (H2O) with only three atoms (shown at the left) to the ribosomal 82. Proteasome 4b4t 96. Single-stranded (Complex I) 3m9s, 3rko DNA-binding Protein 3a5u Water subunits with hundreds of thousands of atoms. 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u 64 83. Ubiquitin 1ubq 21 36. Myoglobin 1mbd 22 87 12 3 4 5 37. Hemoglobin 4hhb 33 Digestive Enzymes: breaking food 68 Fluorescent proteins that are not homologs of GFP into small nutrient moleculeFour classes of fluorescent proteins 69 70 1. Amylase 1smd 5. Pepsin 5pep 6 78 Biliverdin-binding proteins 2. Phospholipase 1poe 6. Trypsin 2ptc Storage: containing nutrients Homologs of 88 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 for future consumption 71 4. Lysozyme 1lz1 8. RibonuAequoreaclease 5rsa GFP with autogenic 38. Ferritin 1hrs 38 chromophore Blood Plasma Proteins: transpformationorting nutrients and defending against injury 34 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf O 72 73 10. Thrombin 1ppb 12. Serum Albumin 1e7i N 11 N 12 14 O O HO 9 N 10 Coral & anemone FPs (e.g. Viruses and : engaging inH Jellyfish FPs (e.g. GFP) mCherry) 89 O O constant battle in the bloodstream NH HN 13. 1igt 14. Rhinovirus 4rhv NH HN Flavin-binding proteins Bilirubin-binding proteins Phytochrome-derived (e.g. IFP1.4, iRFP) 90 HOOC COOH Hormones: carrying molecular 74 messages through blood 15. Glucagon 1gcn 75 16. Insulin 2hiu 15 16 17

17. Epidermal Growth Factor 1egf O O O O NH HN O NH HN N Channels, Pumps and Receptors: gettinNHg NH N NH HN 35 76 91 back and forth across the membraNneN O HOOC COOH 21 HOOC COOH 18. Ras Protein 5p21 R 19. Beta2-Adrenergic Receptor/Gs Protein flavin-binding protein-derived 3sn6 fatty-acid-binding protein-derived Allophycocyanin-derived 20 22 20. Acetylcholine Receptor 2bg9 (e.g. iLOV) (e.g. UnaG) 23 (e.g. smURFP) 24 21. Epidermal Growth Factor Receptor 25 1ivo, 2jwa, 2gs6 19 Shu et al., Science 2009, 324, 804 (IFP1.4); Filonov et al., Nat. Biotechnol. 2011, 29, 757 (iRFP); Yu et al., Nat. Methods 2015, 12, 763 (mIFP); 77 Kumagai et al., Cell 2013, 153, 1602 (UnaG) 22. Rhodopsin 1f88 80 23. P-glycoprotein 4m2t Rodriguez et al. Nat. Methods 2016, 13, 763 (smURFP); Kumagai et al., Cell 2013, 153, 1602 (UnaG); Buckley et al., Curr. Opin. Chem. Biol. 2015, 27, 39 (iLOV). 78 92 24. Potassium Channel 3lut 79 25. Calcium Pump 1su4 26. Cyclooxygenase 1prh 26 18 Photosynthesis: 54 harvesting 53 39 energy from 81 82 the sun Enzymes: cutting and joining the molecules of life 93 55 27. Photosystem II 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1dgk 1s5l 40. RuBisCo: Ribulose Bisphosphate 54. Phosphoglucose Isomerase 1hox 28. Light-harvesting Carboxylase/Oxygenase 1rcx 55. Phosphofructokinase 4pfk 56 Complex 1rwt 41. Green Fluorescent Protein 1gfl 56. Aldolase 4ald 83 29. Photosynthetic 42. Luciferase 2d1s 57. Triosephosphate Isomerase 2ypi 57 93 Reaction 43. Glutamine Synthetase 2gls 58. Glyceraldehyde-3-phosphate 23 Center 1prc 44. Alcohol Dehydrogenase 2ohx Dehydrogenase 3gpd 24 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate Kinase 3pgk 59 94 46. Nitrogenase 1n2c 60. Phospoglycerate Mutase 3pgm Outline 27 Types of fluorophores 58 47. Leucine Aminopeptidase 1lap 61. Enolase 5enl 48. beta-Lactamase 4blm 62. Pyruvate Kinase 1a3w 60 40 49. Catalase 1qqw 66 45 50. Thymidylate Synthase 2tsc 43 51. Tryptophan Synthase 1wsy 51 46 Fluorescent52. Aspartate C arbamoyltransferase 4at1 61 95 28 50 96 Genetically Synthetic 48 encoded 47 Qdot800 52 Fluorescent proteins (FPs) 29 Qdot525 62 41 42 49 44 Scale: Alexa Fluor 488 Cy5 Other fluorophore technologies GFP 1GFL 1nm 5nm 10nm

1nm (nanometer) = e.g., Qdot nanocrystals fromThermoFisher Single FP-based biosensors 10-6 millimeters Extracellular Proteins Membrane Proteins Typically Typically attached toIn tanracell ular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus expressed as antibody directly (dyes) a gene fusion or via streptavidin/ Antibody 1IGT with a protein biotin (Qdots) Streptavidin/biotin 1STP of interest 10 nm C C -B Y -3 .0 D a v i C d C S . -B G Y o -3 o . d 0 s D e a ll v a ng i n di C d d a 4 S 8 C . t -B G h Y e nd re - o R a on 3 o C ti .0 d S ng se B D l e a l P ngs 5 v a D : storinforma di ea 8 id n B a ucl d NA c i n 84 S t D ti do . h ne bna z G e e nd E rne te o R g A 1 aon o N on e 1 6 d C D ngicti rtii s 3 s S 4. tr e lya a i 6 e B 8 es I 1 to e2 8 l . R R o se 1e fa r l P 5 h ang sse 2 e to 85 a D : s8torinEcformA aP dericleaa h 1 n B N oam u er lb va 4 d NA c i D is on 1 8 t D ti 6. po d olym or e Acti h ng ne 8 bnao s z en s e ldi e 7. Tndn ErnAe P res te / ai R g NA 18 a o Nonep e 1 te G n C s icti . Rrtii s li 36 tei IB 1 S . D ngtr 8 e c Rya o a p i o r I B ne 84 es 8I 1 la l ab cg 2 r 86 sis: bui R R 9. oto ta se 1 1 e g Pfa cto P chi 5. 8 h a a sne e 2in e Fa r 5 D : s8tori Eco . C eroeteai as nd 1n toesv u 8 B nforNmAa P90 mPrcl er i bhtio va ta nthe NA c i D iso nu A-b l ip se 4 87 y ar ma x D ti 6. o do lymAT r 1cr e Accati se 1 nga qf6 8 bnaop o. T sonzs eli era in S cul ldi uq ene . T EnA P91 resrtea en aoiais e tna se 1 se 1 e g A 187 on N T A H lymn/ s e1 N cti . R epse 1 liteN6 G o tei e I1B 1 1 5u rote A 4 eta eta s y y . D tri 88 eri c Rya bo. aD3 pAi P ro omr I p5 a P N th nteh eta ff ei 84 es 1 . la l a 2 cgN2 s 1 n 3 86 w mol R sisy:n buiyn th 1 R RI 9 oto atase9 1n 1 De g Pfeaoacto n fer A S chi yn se se 1 5. 8 h C a 3e. 2dinucel F terisv ded otei 8 ne ns N NA S 8 Eco A P 0. er oteia9s in 1on rotoe an u r 8 ra nt-htRe A S eta eta sy N 9 om Pr er 4b. hNti Pva tr ta g P 7 . T y lyl ry l-mtRa N th th a D is TA-b 91l ip U se 4e-s e 1 in 8 67 a an -tR ynax yn e 1 6. po olymA or scr .e H Aicati gl as ind . V reo inyl An Sgg A S qf6 s 8 o 1. T s an 9en5 el n er oi s in68 S cul m lNdei 1 N e 1 etaeuq 7. T A P9 resTr . Syi m /A-b a ai . eTh ta tna-tR s -tR s th 1 ttt 87 N ep te NGA H96 ol Nn e 1 1 u 9 e 6 R i D B 1 5 C rote 6 luA 4 cyl eta ylseta yn s A 1 8. c R ol D p otei m II p5 a th n j2 y N y . A Pr o r C 65 P . G N leu laneth A Seta j ff ei fu 8 la ab92 cg N s 1 n 3 w mo7l0 R o yn a yn N th , 1 1 /tR r e 9. ta 1 g Peocto n Infrastructure: Protein Synthesis: building DNA: storing and reading - ssis: byul i u D a tei B fer . I A S cAh Si -tRyn 5e se T se 1da 1 8 a n 3. in ucl F tei ded o 8 36 ne ns 71 N henN tyl j r 1 Tu . C otei 9 nd n roesv an u r 8 Y ra r A S etacto eta d 90 r i . No P tr ta g P 65 - P e 1 sy ti 3 . T nytlh-tRe2. -tR pa N m th a th r Ga n P A-b94ip Use 4 in 87 supporting and new molecular machines genetic information 7 y l 7 ry ml as o yn F a T e-se 1d . M6 oa lan e-tR scxun yn las r MaAcchr . Hca igl ns n ery: 0 Mole A ca uctole 1ar Macs hi iani ery: V . yl o o a s T 5 n . reo 3 in b A Sgti A Sqf6 r on . T an 9 el er oi in 8S cul 7 i N F cpl 1 r Si A-ba 63 moving cells D 6 e Th m . R se g1a Neo 1n eteaucqto ttt a 9 T A H 6. lymN 30 67. Transfer RNA 4tna 84. DNA 1bna 9. ta tn4a -tR on -tR sti th1 a S 1 e 2 967 N 9 o D e 1 1 u a lu 7 yl yn e F s 5 C rote 6 A 4 c e. tEalnyletgaa osnj2 NA 1L/E a cg . D A P om p5 a 68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease 84 v

y i rcsb.or5 g . GN th5 la thn j iy er 2 s 1 3 C 6 P 0 leyun7 a o A Settai 1 fffxk/tR eoE m fu 0 92 N n 37 d w mol7 R o yn Nthga , 1 r r e 9 eo n 31 63. ActinIn1fmr8aqstructure: Protein Synthesis: building DNA: storing and reading - s yl El 1 u o D A Tour o. f ntehe e Psr 1op tein Da. ta Baednteki EcoRI 1eri B sfer . I A S enA S6 -tRyno j5 s inr Tse G1 da I u s 3 ucl otei d ro 8 36 69. Threonyl-tRNA Synthetase 1qf6 S ne 1 7 d n s 9 n Y n 7 N h N tyl El l i 1 T H r a 8 . r eta n n N a P A. S e 1 ctoeta a d n . P 64. Myosin 1m8q 65 - r . a o sy tr g P G . T yl-tR 2 -tR sp N77 m threfoa tehr r Ga/tr n tei 94 U in supporting and 70. Glutanmeinwyl -mtRNoAle Scynutlhaerta sme1aecuhqines 86. DNAg Pehnoteoltyiacs ei n1fteozrmation 3 l 7 yl F a C e-s . 7 a n -tR o yn P n ynp s s o H d o M6 o. A lse. ca ctuoe 1 lr at r Mac. hgl ininery: 0 o yl o 8 T C 8 5 65. Microtubule1tub

Molecular Machinery: o . V 3 ti a s r n Cells build many complex molecular mach8ines rtehoa7t pineribfoAr 7mS tAh Se Fbioel ocigical jobb4 so nneeded for life. S6ome 9of these machines Energy Production: 71. Isoleucyl-tRNA Synthetase 1ffy 87. Topoisomerase 1a36 n cpl D - Infrastructure: Protein Synthesis: building DNA: storing 6and reamdin Rg N ga N. Chn etlia cto ck P a . Si A-b 0 63 moving cells d Th . n 9 o o a o ttt B 6 N 30 67. Transfer RNA 4tna 84. DNA 1bna 36 : th e 4 9 s . ta 4 -tR o -tR7 ti r S 1 e 2 9 7 9 D 66. Collagen 1bkv a 9 7 P F s 6 Y lu yl yl yn. n t Sh A m1 /E a cg 1 72. Phenylalanyl-tRNA Synthetase 1eiy 88. RNA Polymerase 2e2i e 6 c El ga 7 v 65 are molecular scissors that cut food into digestible pie.cens. Ot0heors2 theno uLse these piec- es to build new molecules when G n j Ns ubqer powering the processes 84 l a 2 3 68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease 5 j ea E i supporting and new molecular machines rcsb5 .goenregtic informatio. n eu l o A 8S ti fxk l

l fu 0 (far left) d 6 0 7 a 1 /tR o 1 m 37 o N H r e 9 . 7 yl El ga, . 1 tea r n o 89. lac Repressor 1lbh 1efa 85 a s . u 0 31 63. Actin 1m8q 73. Aspartyl-tRNA Synthetase 1asy Molecular Machinery: nd A Tour on ef1 n toheti s Pp rotein Data Bank Molecular Machinery: A T oI ur6 ofi tr h G ei 1Ps rotein Data Bank EcoRI 1eri 8 S . -tR o 5 T da I u 69. Threonyl-tRNA Synthetase 1qf6 n a cells grow or tissues need to be repaired. Some 1moleecnu7lar mElacj hilnd er sP fino1rmu ssturdy beams that support cells, and others are of the cell h . q H D moving cells 7 tyl n T . 63 r n i d 30 67. Transtsrfeurc RtuNrAe 4tna 84. DNA 1bna . P a 7. e 1 cto2 o b a d n 32 64. Myosin 1m8q 74. Ribosome 1j5e, 1jj2 90. Cataabolite Gene Activator ng efoa 8 G n G

a 2 p 7 m r er r U /tr tei a 0 86. DNA Photolyase 1tez s s F . s a 70. Glutaminyl-tRNA Synthetase1euq t l 7 o P p o 7 C v s 9 h 68n.f Vralyl-tRNA Synl thetase 1gmaxotors that use energy to c8r5a. wRels atrilcotinogn Ethndeosneu bcleasme s.. A Somose 8r.eocn8o4ganci8tzo3e a Tttacrkerst and mobilize defen8 ses against infection. 30. Cytochrome c Oxidas8e Protein 1cgp o

a i rcsb.org C 65. Microtubule1tub 75. Elongation Factor Tu/tRNA 1ttt e cri e I 3 7 ti 6 o 37 ce Cells build many complex molecular machin7es thiab t perfo Crhm F tinhe bciko Plogbi4caoanl jocbpls ned eded for life. Some of these machines Energy Production: 71. Isoleucyl-tRNA Synthetase 1ffy 87. Topoisomerase 1a36

. l - 31 63. ActinIn1fmr8aqstructure: Protein Synthesis: building DNA: storing and Rreadgaing n cto o (Complex IV) 1oco 0 d 9 o R A Tour of the Protein Data Bank : EcoRI 1eri . n o a e 4 B 9 91. TATA-binding Protein/ 4 7 r e 2 9 S 36 69. sTuhprepoonriytnil-gtRNA Synthetase 1qf6 o ti P F S 1 s 66. Collagen 1bkv 76. Elongation Factor G 1dar s 7 C q m Y 1 . t Sh . ga n o /E a cg 7 88. RNA Polymerase 2e2i e El 0 72. Phenylalanyl-tRNA Synthetase 1eiy 64. Myosin 1m8q 65 q are molecular scissors that cut food into digestib.le pnieceso. Others tLheunb quse these pieces- to build new molecules when31. Cytochrome c 3cyt Transcription Factor IIB 1ais S

ea er G Researchers around the world are study5ing thes8e moleculEes at the a2tomic level. These 3D structures are powering the processes l supporting and new moml8ecular machines 86. DNAg Pehnoteoltyiacs ei n1fteozrmation o ti fxk 3 70. Glutaminyl-tRNA Synthetase1euq 7 o 1m 0 77. Elongation Factor Ts and Tu 1efu l 1 H (far left) B mov ga . tear n 9 . m8 tub El 1 o o nd Adenosine . 1 o ti 89. lac Repressor 1lbh 1efa 86 85 a n n n r i s p 0 73. Aspartyl-tRNA Synthetase 1asy Molecular Machinery: 1 8 i G 65. Microtubule1tub 1 6 I s 32. Cytochrome bc1 92. DNA Helicase 4esv

Molecular Machinery: o o u P a n e 7 d n n Cells build many complex molecular machines that perform the biological jobs needed for life. Some of these machines Energy Production: 71. Isoleucyl-tRNA Si yntheltase 1ffycells grofrwee olyr taisvsauielasb nle87 e.a dTto ttpoho eibs oePm reorptaesaeini r1 eaDd3a.6 tSao mBaen mk o(PleDcEBul )la, rt hml ea .c cPehinintirqeasls fsotrom rHe shtouurdsey boef abmiosm th oalet csulpapr osrttr ucectlulsr, easn. dA o ft3ehwer s are of the cell 67 s n D 78. Prefoldin 1fxk 63 moving cells struc tAuctrie u 2 n d 7 D 30 a 67.3 .Tnragnsfer RNbA 4tnTariphosphate 84. DNA 1bna 7. efo 8 o b a 9n (Complex III) 1bgy 32 74. Ribosome 1j5e, 1jj2 90. Cataabolite Gene Activator d

r s

66. Collagen 1bkv bkv 7 tei 0 a 6 s tu er . U /tr 6 93. DNA Polymerase 1tau l 1 s 7 B

o t 72. Phenylalan Myly-otlRNoA Synthetase 1eiy 88. RNA Polymerase 2e2i . P p 3 r e 9

. (ATP) v 79. Chaperonin GroEL/ES 1aon are molecular scissors that cut food into digestible pieces. Others then use these pieces to build new molecules when nfr cr motors that use energy to crawl along these beams. S8omea re8cognize atttackers and mobilize8 defenses against infection. 30. Cytochrome c8 Oxidase Protein 1cgp h powering the processes 3 I 68. V4alyl-tRNA Synthetase 1gax examples from the ~851.0 R0e,s0tr0ic0tio snt Erundcotunurcelesa shee ld in the P8D4B are shown hel re at a magnification of about 7 e ci i 6 rcsb.org 6 6 l 33. Succinate Dehydrogenase 75. Elongation Factor Tu/tRNA 1ttt e

(far left) ce gen b4 d 37 Mi a . Ch lin ck P 94. Nucleosome 1aoi . l 89. lac Repressor 1lbh 1efa o a 0 31 63. Actin 1m8q 73. Aspartyl-tRNA Synl thetase 1asy 9 85 (Complex IV) 1oco 5 o 9 80. Proline cis/trans Isomerase 2cpl A Tour of the Protein Data Bank ng : EcoRI 1eri 7 r e 4 91. TATA-binding Protein/ R S cells grow or tissues need to be repaired. Some molecular machines form sturdy beams that support cells, and others are of the cell supp6o9ri.t iTh6reon Cyolq-tRNefAt) Synthetase 1qf63,500,000 times, with each atom represented as a Psmat lSlh sphmere. The enorn mous range of molecula1r sizes is (Complex II) 1nek 76. Elongation Factor G 1dar . 95. HU Protein 1p51 C . l . 7 r 0 o d 32 64. Myosin 1m8q 74. Ribosome 1j56e, 1jj2 q 90. Cataabolite Gene Activator 8 ea s ubq 31. Cytochrome c 3cyt 81. Heat Shock Protein Hsp90 2cg9 Transcription Factor IIB 1ais

m8 G 6 S 1 (fa Glucose Researchers aro8u6n. dD NthA eP hwotoorlylads ea 1rete zstudying these mole 1cules at the atomic level. These 3D structures are34. NADH-Quinone Oxidoreductase mo7v0. Glutaminyml-8tRNA Synthetase1euq . H tea n t 77. Elongation Factor Ts and Tu 1efu tub B h motors that use energy to crawl along these beams. Some recognize attackers and mobilize defenses against infection. 30. Cytochrome c Oxidase nnd 1 1 Adenosiniell ustrated here, frPoromte inth 1ec gwp ater molecule (H2O) w81ith roonly ittihree atoms (shown ao t the left) to the ribosomal 1 96. Single-stranded 86 65. Microtubule1tub 75. Elongation Factor Tu/teRNA 1ttt u 32. Cytochrome bc1

n e 82. Proteasome 4b4t a i l P o 3 (Complex I) 3m9s, 3rko 9 92. DNA Helicase 4esv Cells build many complex molecular machines that perform the biological jobs needed for life. Some of these machines Energy Production: 71. I sAoctlieucsyl-tRNuA Synthetase 1ffy 87. Topoisomerase 1a36 . iq 7 67 P struc.ture freely available at the Protein Data Bank (PDB2), the central storeho use of biomolecular structures. A few 78. Prefoldin 1fxk DNA-binding Protein 3a5u (Complex IV) 1oco b b d

Triphosphate 8 9 D a 63 ng tu bWkvater 91. TATA-binding Protein/ 6 R 0 (Complex III) 1bgy 64 66. Collagen 1bkv 76. Elongation Falcstor G 1d1ar subunits with hundreds of thousands of atoms. . U s 83. Ubiquitin 1ubq 93. DNA Polymerase 1tau o C 7 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u Mylo 9 B are molecular scissors that cut food into digestible pieces. Others then use these pieces to build new molecules when31. Cytochrome c 3cyt 3 nfr 72. P4h. enylcarlanyl-tRNA Syn(tAhTePta)se 1eiy Trans8c8ri.p RtiNonA FPaocltyomr eIIrBa s1ea 2ise2i 83 e 8 2 79. Chaperonin GroEL/ES 1aon S Researchers around the world are studying these molecules at the atomic level. These 3D structures are powering the processes 6 I 6 gen examples from the ~100,000 structures held in the PDB are shown herl e at a magnification of about 9 77. Elongation c Fe aMcitor Ts and Tu 1efu l 33. Succinate Dehydrogenase (far left) a B . ll 36. Myoglobin 1mbd 94. Nucleosome 1aoi Adenosine 73. As5partyl-tRNA Synthetase 1asy 89. lac Repressor 1lbh 1efa 86 85 a 4 32. Cytochrome bc1 supporitnig6 92. DNA Helicase 4esv 7 80. Proline cis/trans Isomerase 2cpl 87 P eft) n (Complex II) 1nek cells grofrwee olyr taisvsauielasb nle eadt ttoh eb eP roepteainir eDda. tSao mBaen mk o(PleDcBu)la, rt hmea ccehnintreasl fsotromre shtouurdsey boef abmiosm thoalet csulpapr osrttr ucectlulsr, easn. dA o ftehwer s are of the cell 6 78. Prefoldin 1fx6k. Coq r l 67 3,500,000 times, with ea12ch atom represen 3ted as a sm 4all sphere. T5he enormous range of molecular sizes is 95. HU Protein 1p51 D Triphosphate 2 d 37. Hemoglobin 4hhb (Complex III) 1bgy 32 3 74. Ribo6smom8(efa 1jq5e, 1jj2 90. Cataabolite Gene Activator 7 33 81. Heat Shock Protein Hsp90 2cg9 1 93. DNA Polymerase 1tau mov m8 Glucose B 34. NADH-Quinone Oxidoreductase (ATP) 79. Chaperoninn GroELD/EiSg 1teuabsotnive Enzymes: breaking food t 30. Cytochrome c Oxidase 1 h 96. Single-stranded motorse txhaamt upsles e fnreormgyt thoe c ~ra1w0l 0a,l0o0n0g sthtreuscet ubreeasm hse. lSdo mine t hreec oPgDnBiz ea raett aschkoewrsn a hnedr me oabt iali zme daegfneinfisceast iaogna ionfs ta ibnofeucttion.33. Succinate Dehydrogenase 75. Elongainti o1n Flaector Tu/tRNA 1ttt illustrated here, froPmro ttehine 1 wcgapter molecule (H2O) with only three atoms (shown at the left) to the r3ibosomal 91 68 . Acti s u 94. Nucleosome 1aoi e 7 (Complex I) 3m9s, 3rko 82. Proteasome 4b4t (Complex IV) 1oco 80. Proline3 cis/trans Isobmerase 2cpl 9 DNA-binding Protein 3a5u

bkv 91. TATA-binding Protein/ R 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is (Complex II) 1nek 766. El oMnyogatoiotuni nFat1coto sr mG 1adlWla arnteurtrient msuobluencuitsle wsith hundreds of thousands of atoms. 6 64 83. Ubiquitin 1ubq . 95. HU Protein 1p51 C 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u 70 31. Cytochrome c 3cyt 7 63 81. Heat Sh6o4ck Procrtein Hsp90 2cg9 Transcription Factor IIB 1ais 92 69 3 S Glucose Researchers around the world are studying these molecules at the atomic level. These 3D structures are34. NADH-Quinone Oxidoreductase 77. Elo.n Mgiatiolang1 e.FnaActmory lTass ea n1ds mTud 1efu 64 5. Pepsin 5pep Adenosiniell ustrated here, from the water molecule (H2O) with only three atoms (shown at the left) to the ribosomal 65 l 96. Single-stranded 6 86 78B 36.74Myoglobin 1mbd (Co3m2p. Cleyxt oI)c h3rmom9se, 3brck1o 82. Proteasome 4 Cbo4t eft) 92. DNA Helicase 4esv 87 freely available at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. A few 36 78. Pref6o.ldin 1r2 fl.xkPhospholipase 1poe 667. Trypsin 2pDtNc A-binding Protein12 3a5u 3 4 5 2 P Storage: containing nutrients

Triphosphate 7 D 37. Hemoglobin 4hhb Water subunits with hundreds of thousands of atoms. 35. ATP Syn(Cthoamsep 1leex7 I9II,) 1 1cb17gy, 1l2p, 2a7u 64 83. Ubiquitin 16ubq (fa 93. DNA Polymerase 1tau 33 80 88 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa B 13 (ATP) examples from the ~100,000 structures held in the PDB are shown here at a magnification of about 79. Chaperonin GroEDLi/gESe 1satoivne Enzymes: breaking food for future consumption 91 68 71 36. Myo3g3l.oSbuinccin1amteb dDehydrogenase 4. Lysozyme 1lz1 8. Ribonuclease 59r4s.a Nucleosome 1aoi 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is (Complex II) 1nek 80. Proline cis/trans iInsotmoe sramsea 2lcl pnl utrient molecules 87 38. Ferritin 1hrs 38 12 3 4 5 37. Hemoglobin 4hhb 0 37 95. HU Protein 1p51 92 70 Glucose 34. NADH-Quinone Oxidoreductas3e3 3 81. Heat Shock Protein Hsp90 2cg9 64 69 Digestive Enzymesi:llbusrterakteindg h feoreo,d from the water molecule (H2O) with only three atoms (shown at the left) to the ribosomal 1. Amylase 1smd 5. Pepsin 59p6e. pSingle-stranded 6 78 74 3 6 82. Proteasome 4b4t 68 9 (Complex I) 3m9s, 3rko 3 Bloo2d. PPhloaspshmolaip aPser o1ptoeeins: tr6a. nTrsyppsoinr t2ipntgcD NnAu-tbriniednintgs Protein 3a5u 72 0 into smalWl anteurtrient molecules 64 75 Storage: containing nutrients 8 88 subunits with hundreds of thousands of atoms. 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u 83. Ubiquitin 1u7b0q 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 93 1. Amylase 1smd 5. Pepsin 5pep 69 and defending against injury for future consumption 9 71 6 78 36. Myoglobin 1mbd 4. Lysozyme 1lz1 8. Ribonuclease 5rsa 34 7 2. Phospholipase 1poe 6. Trypsin 2ptc 1 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf 87 38 12 3 4 5 Stora3g7.eH:ecmoongtloabiinn i4nhgh bnutrients 3 0 37 38. Ferritin 1hrs 72 73 33 : 3 64 88 3. DeoDxiygribeosntuicvleea sEe n2dznyjm7e. sC: abrbroexaypkeinptgid afsoeo 3dcpa 13 10. Thrombin 1ppb 712. Serum Albumin 1e7i 3 for future consumption s 2 68 11 12 9 4. Lysozyme 1lz1 8. Ribonuclease 5rsa y 3 14 5 0 into small nutrient molecules 38 sse Blood Plasma Proteins: transporting nutrients 7 76 8 3 38. Ferritin 1hrs r 70 9 9 69 and defending against injury 10 78 79 1. Amylase 1smd 5. Pepsin 5pep 6 78 e roduc tpioronc: e Viruses and Antibodies: engaging in 34 89 P 1 Bloo2d. PPhloaspshmolaip aPser o1ptoeeins: tr6a. nTrsyppsoinr t2ipntgc nutrients Storage: containing nutrients y 3 0 e 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf : rg the 3 as constant battle in the bloodstream 88 72 73 n d n ng i 3 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 k 10. Thrombin 1ppb 12. Serum Albumin 1e7i 77 9 and defending against injury for future consumption i ne ri l s 32 13. Antibody 1igt 14. Rhi7n1ovirus 4rhv 34 i y E l 11 12 n y 5 n we e c Oxoco 14 6 6 4. Lysozyme 1lz1 8. Ribonuclease 5rsa ce m sse t 7 7 3 9 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf 38. Ferritin 1hrs 38 ar po ro V) 1 cy 9 h r s 73 90 I 9 8 B ne ch ex 72 10 7 79 10. Thrombin 1ppb 12. Serum Albumin 1e7i c B hi of therotoducp tlpioronc: e c 3 se Viruses and Antibodies: engaging in e n c1 y a e 89 e c Cy m 11 P 1 m o s Hormones: carrying molecular 12 14 a a y0. 3 r e be bg 4 Blood Plasma Proteins: transporting nutrients a m whe e 3 (C tohe ch m as) 1 ogen ucta 7u 8 83 9 74 a t rg I r t : to o a constant battle in the bloodstream n e : r r d I 3 n s ng i 9 s ke a I yd red 2 messages through blood 77 10 ai e s ne ri . Cly ch lex s eh k 2 o p, i h r l to d Viruasensd adnedfe Andnitnibgo adgiaeisn:set ningjaugrying in t nculye E 31 p ne 3 i l2 33 13. Antibody 1igt 14. Rhinovirus 4rhv f ny 6 34 e h we Cy e mc Oxoco rko 1 15. Glucagon 1gcn 89 6 M D o t on. ce2. m ) 1ssatee yDt I) 1 e Ox 3 7, 7 9 M a o I 75 h e a s po 3 ro(CoV n c x n s, 2 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf r cti c1 8 h r 17 5 constant battle in the bloodstream m mol d e ch x I cci le no 1 16. Insulin 2hiu 73 15 16 8 9 n 7 90 n n fe e p i m9 , 72 nc So Bw , a hiin aroef threotdoucptiloSnu: e mc 3 se7 9 i c p.roce 10. Thrombin 1ppb 12. Serum Albumin 1e7i i . es c t n Cy 3 m c1-Qu y ) 3 a e e ne e m r l I s r s Co 17. Epidermal Growth Factor 1egf s o 13. Antibody 1igt 14. Rhinovirus 4rhv 11 12 e ife ad el an yw P0. 3 r ( e bH bgex e 1 Hormones: carrying molecular 4 14 a r l l c mai whe e fe3 ( Ctho e ch mADse) 1pl ogaesn ucta 7u 8 83 9 t a t t a I r 74 a t o tnr e g s r rg t to roNd I m th mbd a 3 a s i 1 ed buin ng . I f o e a ke a A u Cy ch4 x yd k r 2 77 9 l o d a p h s rs ns.e roi . l to 3 le (Co eh yn n do hhb p, messages through blood 90 1 C to i i l e l 2 3 8 i 1 i 10 e p t E p S ne nts cul setructuree b 3 b 4 l C Viruses and Antibodies: engaging in r d s su fns ne h r wae is Cy e cm Oxoco TP ) 1 o in rko , 1 3 e 6 e cDe t oe Dt tuon. f ce2. m ateA Dt I gl e Oxb 3 7 Chan15n.eGllsu,c aPguonm 1pgcsn and Receptors: getting 89 9 - Hormones: carrying molecular M s B M e f o ) 1 . y I o ding u P n e a ee a 3 cti poo 3 ro (CoV n5c x n s, c1 82 35 DNA: storing and rea h s 5 u gl lding h 75 Y s pi th md mol d eru ze l I cci3 le Myono o 1 74 9 Protein Synthesis: bui 76 n a x se fe n ch p i , 17 constant battle in the bloodstream b n s oe Bw a insnt re si le . m9 9 nutri 16. Insulin 2hiu 15 16 nfrastructure: - 91 messages through blood c e o ise Sz , i io aorf the tom p . Su e cm 3 6 em se 7 back and forth across the membrane I 3 c h 3 Qu c e j i c i he r t ) 3 m . s t a o c1 y e ng nformation r l ne l c n Cy 3 m - H I a e genetic i . r l i T as a s s m Co 21 65 a e l s on chines 0 13. Antibody 1igt 14. Rhinovirus 4rhv h ethe e lif b ad l.e auiln ic cul w0.o 3 ro ( e bH 7b. glex e 1 ini 17. Epidermal Growth Factor 1egf 36 4 new molecular ma h ica b o al e c mc if e3b (Co ch AD 3 p gesn cta u 18. Ras Protein 5p21 ng and3 9 15. Glucagon 1gcn e t a r t a whe e e f i m ) 1 o a u 7 8 supporti 8 D e t g t r r a v I ta y o m t r eleg n r t to ro N I m th mbd a 3 a o s 1 f bui e s a g A . ed 75 l l use rd d lo o e a e u Cy ch 4 x I yd k r 2 34 84. DNA 1bna a 17 l o e a . yn 16 o o d a p s s mol h 3 e (Co o hhb p, 19. Beta2-Adrenergic Receptor/Gs Protein 3sn6 1 RNA 4tna 16. Insulin 2hiu 15 f l i n u n c s t o . l eh n conta 67. Transfer v f l to hme r i d 8 b t ea p t m structuree b 1 to p S ne b i 4 l2 3 nts : 90 moving cells s : 20 y i cul e P r 63 r s o : 3 e s 3 y 22 4 u n 8 r of o r e i d r s n e i h f c n d o i a a Cy T i h ease s t i c onucl n e M d h m e En rko a n s 1 r ictio e a . Restr M 5 the a l 8 o e ) 1 r u , u D b t . te D c a r o D A l 23 o b e D gl l u e on. I c e o x t f e 0 e 1ga ce t ) l 3 etas M th m s g 20. Acetylcholine Receptor 2bg9 yn o 17. Epidermal Growth Factor 1egf h e tomi f 2 a . e Ox 3 7 consumpti M . Valyl-tRNA S o 68 M e o o t M f t 3o t I P r e a c 2 Hormones: carrying molecular u Prs n k a e e f o 3 (Co n 5 x n s, a Channels, Pumps and Receptors: getting S u a cti gl c1 8 fo c h do m ol dng ule ze l 3 le o o 5 EcoRI 1eri r m s pi t m a r n a cci Myon 1 24 35 9 . r sne tfe si nutri us r s b ta n s oe wr e r s e o re p . i m9tor , 21. Epidermal Growth Factor Recep7t4or eonyl-tRNA Synthetase 1qef6 76 c o e e o t se the S sz a in i r m Su m 6 em 79 hrs 37 25 63. Actin 1m8q 69. Thr G 91 c f e j i i e h,e h t a . 3 S u r Othe in a i t m . iul ne Ts tr t a a o 3 -QuH ) 3 e 1 ng back and forth across the membrane NA Photolyase 1tez messages through blood r l h l a s I on 86. D o r a o s Co n 31 M e s. h e the ae fehb . l lst ic o 3 ( H 7. x ti ni .org 1ivo, 2jwa, 2gs6 19 ynthetase1euq M h z e i d le una w e i i rcsb nk 21 minyl-tRNA S o p h c icai e sb leo l n c i f cul e b 3 l se 1 r tein Data Bank 64. Myosin 1m8q 70. Gluta 77 o oe t ce a n e r e can i e f i AD p a for future of the Protein ns: Nucl t t r er ur th i e g w t r o f d t t y m v e n mbd A Tour o poisomerase 1a36 Chan15n.eGllsu,c aPguonm 1pgcsn and Receptors: getting a e a o g oo er lg t N m th F A 18. Ras Protein 5p21 87. To T am t o w g 1 4 T h l use d f bui l a A e . . 22. Rhodopsin 1f88 35 l t pi o crul sd onormo us u 4 3 . Isoleucyl-tRNA Synthetase 1ffy s l r h o a 8 crotubule1tub 71 rote o c o s mol . h yn 75 65. Mi 80 s f l a i e on eu dln s cp e mh s t o 3 (Co n 3hhb conta 76 1 e e l l t to e i 8 16 17 br eaa p s m S 4 : nts 91 r P 88. RNA Polymerase 2e2i 16. Insulin 2hiu 15 e u r s r s e u ios( storuf ctureeo b P b 19. Beta2-Adrenergic Receptor/Gs Protein 3sn6 a l back and forth across the membrane n bl c e e the dts r he n a r t a is T o in e e 23. P-glycoprotein 4m2t -tRNA Synthetase 1eiy l A i o mmol enr a s bs D e u) A gl b g 20 chines Energy Production22: 66. Collagen 1bkv 72. Phenylalanyl lul 21 o e h ce ttomT i e t f s consumpti of these ma l a o h sti l e e t t . life. Some c e uo t m Prs r n ek e aa. effm a 3 c f o 5 o a that perform20 .tAhcee tbylicohlooligneic Rael cjeopbtosr n2ebeg9ded for 23 89. lac Repressor 1lbh 1efa 78 85 92 17. Epidermal Growth Factor 1egf u o o c o ng gl achines ce n c B u e ze l 3 molecular m 18. Ras Protein 5p21 a rg m m S s fe s a pDi thre e dto e se a r l n a Myo o Cells build many complex 24. Potassium Channel 3lut C 73. Aspartyl-tRNa A Synthetase 1asyus m r . s b et a r r t e si . tor nutri (far left) e 79 d g di e o s thee ht P thse zse s io r m 6 em hrs ecules when powering the processes 24 C Intr ene Activator r c f e ot se u e h S ld new mol ataabolite G c eOthe j a i e he t 3 21. Epidermal Growth Factor Recepcteosr to bui 90. C r u n tm r t a o 1 ng n use these pi u r - 19. Beta2-Adrenergic Receptor/Gs Protein 3sn6 la m m i l , e a oile h T a t a s H n on Infrastructure: into digestible p2ie5.cCeasl.c iOumth Peurms pth 1esu4 Protein Synthesis: building DNA: storing and reading 25 1jj2 t e e, h M nto o ea s. h ) h a sphehb r . l c 36 . tmi olenicular scissors that cut food B 74. Ribosome 1j5 o Mu ng z el a o 7 arei 20 i S pe h c caBi thte s l h n l u i cul 3 3 r i 19 cgp 22 o e f n 1 e . ec oe. t b ce a iDn e b r o t w e c5n i e ib 1 for futeur re 1ivo, 2jwa, 2gs6 thers are of the cell Y ins: Nucl Protei t g n r 65 ells, and o 23 e v rt c - 20. Acetylcholine Receptor 2bg9 b l T d a te m Pg i y o mly o lew n F 26. Cyclooxygineneass efo 1rpmrh sturdy beams that suppo 32 tion Factor Tu/tRNA 1ttt 77 3 ga Channels, Pumps and Receptors: getting o Tl re thse pi r lo c(o us e crudslma sdtn h lenormooous g e 8. r tissues unpepedo rtoti nbge 3ra4enpda ired. Some molecular mach new molecular machines genetic information 75. Elon rote tein/ R s foodl d a cells grow o TA-binding Pro o mol h . 91. TA 24 m i s e f satudyii ke n l eu lno s c e h t 3 conta 22. Rhodopsin 1f88 C Mole f 0 cular e M m a 35 ch iner y a l : Mo lec e ul s a a 26 r M a a d ch i inery: t 21. Epidermal Growth Factor Receptor c x eth u bn r e s aah e o (s m : 76 infection. 30. Cytochrome c Ox r PG 1dar 80 p s r of o ses against ongation Factoa S 25r le Acut in bl rce e ae h tsit r he i t e gnize attackers and mobilize defen 91 76. El iption Factor IIB 1ais A e g o the a r b s a 63 moving cells ams. Some reco 18 D ul Transcr back and forth across the membrane p t o r hn soti ale h mB s mol enw a to mTi 30 t e t) g to crosn tshuamt putsie energy to crawl along these be 23. P-glycoprotein 4m2t 67. Transfer RNA 4tna lex IV) 18o4c.o DNA 1bna ll B 1ivo, 2jwa, 2gs6 19 c e t e r d e . f m f a mo (Comp 86 e a rs a 21 m a lo do to r soe ck) B a o o a ng e . Elongation Fceactor Ts and Tu 1efu tha b rg maS u f c re t a l 77 54 77 a case 4esv 78 P 92 co m a dri m r . t s nte tea O D e a e r Photosynthesis: v eus 92. DNA Heli 18. Ras Protein 5p21 rcsb.org g o l D h 2 P s r e tor 24. Potassium Channel 3lut 68. Valyl-tRNA Synthetase 1gax 85cy. Rt estriction Endonuclease 84 D 22. Rhodopsin 1f88 t r e o s c tehe t t the hrs 31. Cytochrome c 3 i Intr 67 y r a w u worl rfn Outhe n se aH t u e e th 37 S 1 D structures are d refoldin 1fxk 79 A Tour of the Protn ein Dd laa ta Bm ainm kr i , ( e sp3h1e o e h n 63. Actin 1m8q s at the atomic level. These8 03 78. P ase 1tau B A Tour of the Proatoein eDuar tnato Bo aeeansk.t nhg )e h a h r t ti d are studying these molecule EcoRI 1eri 53 93. DNA Polymer ssoMrs e . l worl S M c i s Bz t h a i round the 25. Calcium Pump 1su4 23. P-g1l9y.coBpertoa2te-iAnd 4remn2etrgic Receptor/Gs Protein 3sn6 m . ne c o t. hSe pte cpre i s s l e t n 13 r Researchers a harvesting 69. Threonyl-tRNA Synthetase 1qf6 32. Cytochrome bc1 ns: Nucl 20 sci le o t o b c0e a Dn n e r w 5n78 for futeur re . aperonin GroEL/ESi 1aon 22 d b s t d aer e0 re Pgcul i m o ly enosine 64. Myosin 1m8q 39 uctures. A few 79. Ch some 1aoi 23 l r e o T e hP m (oe o t n o w . AF d 78 orehouse of biomolecular str 86. yDNA Photolyase 1tez G 94. Nucleo 24. Po2ta0s.sAiucmet yClchhaonlnineel 3Rleucteptor 2bg9 ui s a u mgy food ir tee s p,0i strudyi kc ldat cu slma so sh enormoous 38 ailable at the Protein Data Bank (PD2B6.),C ythcloeo cxyegnetnraasle s1tprh 70. Glutaminyl-tRNA Synthetase1euq (Complex9 2III) 1bg rote 81 82 e l ely av o 24 b c iss x er rpoaunde tshth 0e0 latom nrme ol e f s a a h (sh Triphosphate fre 65. Microtubule1tub energy from 79 80. Proline cis/transr IPsomerase 2cpl n 1p51 cul u a ho r t e o a 95. HU Protei 21. Epidermal Growth Factor Receptor Cells build many complex molecular machines that perform the biologicallsl jorbse need etd folern lifeA. cSuot mraee of itnhtg ese mb1lachaircneaes Ber En es rgmyo alProtdiuctiron: he s 4 shown here 7a1.t Isao lemucaygl-tnRiNfiAc aSytinothne taosfe a1fbfyout 33. Succinate Dehy8d7.r oTogepnoaissoem erase 1a36 26 ul 25 r d 25. Calcium Pump 1su4 e o pe e t res cho an es ~ti d lech am te esd d en) w a . T m 1 (ATP) examples from the ~100,000 structures held in the PDB are 18 Enzymes: cutting and joining the molecules of life 81. Heat Shock Pllrotein Hsp90 2cg9 93 1ivo, 2jwa, 2gs6 19 C mol ms tha b alel ge oa to r n se c B 6o 66. Collagen 1bkv th5 e sun (Complex II) 18n8e.k RNA Polymerase 2e2i s ace 96. Single-stranded 26. Cyclooxygenase 1prh are molecular scissors that cut food into digestible pieces. Others then use theosew p iceo cgues to buiold nemwbl a modtilhecule mse whD.e a Swna tuspaowneterineg2O theD procreesseast 77 3 72. Prahenngyela loanf yml-tRoNleAc Suylnatrh estiazsees 1 iesiy e 55 re r t r cthe la e s cu the h P small sphere. The enormous l 54 teasInotmr e 4b4t 5u g y ar riw wothr l u se t H e (far left) each atom representedP ahso atosynthesis: 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1l dgk 82. Pro DNA-binding Protein 3a 22. Rhodopsin 1f88 a nh ad aaa m inm r o , ( e e 3,500,000 times, with 27. Photosystem II 34. NADH-Quinon8e9 O. lxaicd oRreepdruescstaosre 1 lbh 1efa l m the t sphe 85 a cells grow or tissues need to be repaired. Some molecul lls a ot ssors seee26uacvr inoto o wi tea s th ngpre B) e th . l hr 3 73. Aspartyl-tRNA Synt8h0etase 1asy ar machines form sturdy bseams that suppo rt cells, anSd otheers ar e s l mal n 18 ce mr. sci n eyc o fr .t hse, r ob 0 of re of Dthe cne ll t 578 1 Glucose 1Hs25lO) with only three atoms (shown at the left) to the riboso (Complex I) 3m9s, 3rko 40. RuBisCo: Ribulose Bisphosphate 54. Phosphoglucose Isomeras5e3 1hox 64 83. Ubiquitin 1ubq Re t m ( d 23. P-glycoprotein 4m2t ildtob r es oelyl es ed Pefrom 0s (Pecul ito ly 32 illustrated here, from the water molecule harvesting 74. Ribosome 1j5e, 1jj2 90. Cataabolite Gene Activator os a u eg folod r me es ,0d 12d sma 3n 4 39 56 87 motors that use energy to crawl along these beams. Some recognize attackeurs and mobs ilize m rdr efenpses aig tai inste i,nfec0tieons5.to4umdy i k30. Cylto achroame c O xoidase 2 78 e 1e792, 1c17, 1l2p, C2ar7buoxylase/Oxygenase 1rcx 55. Phosphofruc t okinase 4pfk s h Phot2o4s. yPontatshseiusmis C: hannel 3lut b mc cul tis x fe m rpoa0und tthhre 0dr at anmol heof s th 1 ands of a2to8.mLisg.ht-harvesting 75. Elongation Factor Tu/tRNA 1ttt 35. ATP Syntha Protein 1cgp 81 82 s s e ll r e r ne leen t caut rae00 atghe 1n are B r s (sC ompalex IV) i1oco 14 Water subunits with hundreds of thou energy from 79 41. Green Fluorescent Protein 1gfl e o si p te x , n ~u ch te d d w Complex 1rwt globin 1mbd 56. Aldolase 4ald 83 ea e rs e 36. Myo 91. TATA-binding Protein/ R harve25s.tiCnaglc ium Pump 1su4 C mol o msh 53 b0e0 lelod e h d a ta ran ) 6 35 76. Elongation Factor G 1dar Enzymes: cutting and joining the molecules of life 93 o up tha ba h e a u O C 93 39 Researchers around the world are studying these moleculesre at threow aetn ocmto s ic level. toT,h5 eseal a3teD sthtritucturesD a rwea ctus31. Cnytteochr2ome c 3cyt 29. Phtohtoes ysnuthnetic 4hhbTranscriptio4n2 .FLaucctoifre rIaIBse 1 2adis1s 57. Triosephosphate Isomerase 2ypi 57 68 26. Cyclooxygenase 1prh a g Ady ah adr c3he aiwl w wothrl n u o se (H 82 5 77. Elongation Factor Ts and Tu 1efu 37. Hemoglobin 33 S 55 Adenosine n hp a m i r 81 12 3 4 B energy from lls a T tri s)sors see vrustr oits wi e the st th 32. Cyteo chrom. e bc1 Reaction 43. Glu3ta9m. Fiantety S Ayncitdh eStyanseth 2asgels 2uvb8,6 2uvc 53. Hexokinase 1dgk tosol e s P e a cll r thse, t fofod pre s 27. Photosystem II 92. DNA Helicase 4esv 58. Glyceraldehy de-3-phosphate y Triphosphate freely available at the Protein Data Bank (PDB), the central sctore mhoorusseTc iof bsR ineomolytloeci ul2as6 rfu sntructurreos. A f0e wo re cul m 78 food 748. Prefoldin 1fxk 67 P 70 Enzymes: cutting and joining the mldotlecur( Ales of life b P n0gs e(Compolex III) 1bgy estive Enzymes: breaking Center 1prc 5 93 40. RuBisCo: Ribulose Bisphosphate 54. PhospD hoglucose Isomerase 1hox the sun 18 i o a ue egey leu time e , fromk,i0 d 12t 3 4 2 Dig 1s5l 93. DNA Polym44e.raAsleco 1htoalu Dehydrogenase 2ohx Dehydrogenase 3gpd ins: C 69 u e a B (ATP) examples from the ~100,000 structures held in the PDB abrem shown hssere saeferrt a mpasgni0f5i 5catthioren oaf0 arbosutotm p f cpa 1 79. Chaperonin GroEL/ES 1aon 56 Photosynthesis: ls ecul ti e con am aro0undt 10nd ea pre m53o43l. S uoccinate Dehydrogenase 4 ent molecules 28. Light-harvesting 45. DihydroCfaorlbaotex yRlaesdeu/cOtaxsyege1ndahsfe 1rcx 59. Pho5s5p.h Pohgolyscpehroatferu Kcitnoaksinea 3sep g4kpfk rote 27. Photosystem II 39. Fatty Acid Synthase 2uvb, 2uvc el 53. Hexokinarssein 1dlu getek x ,0 a he ~breu 5 ds se 3 1 1 into small nutri 94. Nucleosome 1aoi 59 r P 88 94 C o o Ghea e rs0 e d h accuhl sin te ptcn d(Ca omplex II) 1nek 1 5 80. Proline cis/trans Isomerase 2cpl 41. Green Fluorescent Protein 1gfl ula 3,500,000 times, with each40 a. tRoumBis Creo:p Rriebsuelonsete Bdis pahso asp shmatea ll sphe5r 4me..Po Thl ohsep heeongoluurscpsmosoe uIsso mraenrag5s5e03 1ohbfo lmxteolesce:tuhlar esizes wisa 2 a ti nts 6 ase 1smd 5. Pepsin 5pep 3 Comp6lex 1rwt 78 95. HU Protein461.pN5i1trogenase 1n2c 60. Pho5s6p.oAglldyocelarsaete 4 Maludtase 3pgm ll 71 83 1s5hlarvesting re row to ter rch,e la a thi Pep sin us ep rsa e 1. Amyl 5 81. H Storage: containing nutrients ce 39 Glucose a g Ad pah Wa a 3 ai m ew th . 5o6 yp34. NeA 5DH-Quinone Oxidoreductase 2 29. Photosynthe2t7ic eat Shock Protein Hsp90 2cg9 42. Luciferase 2d1s 57. Triosephosphate Isomerase 528ypi 57 a 93 28. Light-harvesting illustrated here, from the waCtearb omxyolalesec/uOlxey g(eHn2asOe )1 rwcxith only th5r5el.el sP haotospm hTtrhosi f r(usc)htookwinnsaes ea t4 pthfkve ulsetfrt)o ymtiots the w riibo5stohmeTrayplth ox s 2. Phospholipase 1poe 6. Trypsin 2ptc 82 96. Single-stra4n7d.eLde ucine Aminopeptidase 1lap 61. Enolase 5enl Intr tosol energy from e rs TP aill nfrzn sn,t mol . fof odrb clea(Complex I) n3umtr9is, 3rko 81 6 82. Proteasome 4b134t on y c e o Reaction 4 consumpti 43. Glutamine Synthetase 2gls C Complex 1rwt 41. Green Fluorescent Protein 1gfl 56. Aldtoolase( A4ald Re ly sE bu n6gs Ca nu uclease 2dnj 7. Carboxypeptidase 3cpa 2 83 5 for future DNA-bindi4n8g. Pbreottae-iLna c3ta5muase 4blm 62. Pyru5v8a.tGe lKycinearasled e1hay3dwe-3-phosphate e e me from ng 3. Deoxyribon 64 ns: C C Water subunits with hundreds of thousEandzsy mof easto:mcus.tting and joining tohe moleculeese of lifeles u ti , kied 7. bo 3125. ATP Synthase 1e 379, 1c17, 1 4l2p, 2a7u 2 Center 1prc 83. Ubiquitin 1u5b6q 93 38 60 i the sun m sfr p a r s i p cpa 0 1 93 44. Alcohol Dehydrogenase 2ohx Dehydrogenase 3gpd Infrastructure: Protein Synthesis: building DNA: storing and reading - R ritin 1hrs B 29. Photosynthetic 42. Luciferase 2d1s 57. Triosephospeh ateco Isomesrmtaisve 2lyl0 pn0iutri re 57 d j e . pe 1 4. Lysozyme 1lz1 8. Ribonuclease 5rsa 40 38. Fer 49. Catalase 1qqw rote 36 in lute exa ,0 he 5pbo5eruen n 8 5 36. Msyeo 3globin 1mbd 1 r P Y Reaction 39. Fatty Acid Synthase 2uvb, 2uvc 53. HesxokGianase 1edgk 0 smd h dcul in ptc a 1 66 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate Kinase 3pgk a 65 94 - 43. Glutamine Synthetase 2gls o 2 nsporti 45 3 27. Photosystem II 58. Glyceraldehpyhde-3-phosph0ate d es 1:h e e s 2 taid f ni ts 3 50. Thymidylate Synthase 2tsc 87 59llul supporting and new molecular machines genetic information 12 3 4 en5 s Dig 5 e 1te s t s ep n rsa ba 7 5 . ter , s a a i a P i 3e7tp.r Hemoglobin 4hhebe 9 5 Molecular Machinery: ce 0 Center 1prc o p s 2 5 Molecular Machine60. Phrospoyglycer:ate Mutase 3pgm 1s5l 40. RuBisCo: Ribulose Bisphosphate 5A4d. Phhosphoglucose3 Isomaeraseim 1ehoxle , 5 46. Nitrogenase 1n2c a 44. Alcohol Dehydrogenase 2ohx Dehydropgenase W3agpd yl l w c . yp yp e y5 j 1 33 2 43 51. Tryptophan Synthase 1wsy 51 Digestive Enzymes: breaking food i nto sma oy s u 5 r x s in 63 moving cells D Carboxylase/Oxygenase 1rcx Tr P) i Amlustrph it nnt mol T bions: e5au6r m1 m lasma Protein4s: transporting nutrients 27 58 30 Intr tosol 67. Transfer RNA 4tna 84. DNA 1bna l nz o food Blood P 2 6 68 47. Leucine Aminopeptidase 1lap 61. Enolase 5enl y a 28. Light-harvesting 45. Dihydrofolate Reductase 1dhf 59. Pho5s5p.h PohgTolyscpehroatferu Kcitnoak.sineai 3sep go4skpufnk ib e lz1 6. ar uclnj n 1 bu nutri 2 4 94 46 52. Aspartate Carbamoyltransferase 4at1 (A 1 Ph Eb yr ng C 5n9 ri ng ife hox 5 7 9 34 73 68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease 84 v into small nutrient molecules eu x e 1 . o ib Al rcsb.o5 rg 6 5 ins: C 72 i 41. Green Fluorescent Protein 1gfl ki rote d Complex 1rwt 46. Nitrogenase 1n2c 60. Pho5s6p.oAglldyocelarsaete 4 sMaeludtase2 .3psgmeo a 7 Ribnpst i F m in 0 and defending against injury e 1 83 5 48. beta-Lactamase 4blm 62. Pyruvate Kinase 1a3w 31 61 37 63. Actin 1m8q 95 ym Ps . cupa 1 s 7A0 Tour of the Protein Data Bank 60 D nuztri i 1 A Tour of the Protein Data Bank rote EcoRI 1eri 27 1. Amylase 1smd 5. Pepsin 5pep 42. Luciferase 2d1s uco estiv. ll o 58 oe nj e57 8. ape 1 eS 3er ng 23 s2 8of l era 69 93 69. Threonyl-tRNA Synthetase 1qf6 S 29. Photosynthetic 6 7857. Triosepl hosphate Is3omeryasse 2ypipbre d g 5 s. i 1 . Fibrin 1m1j, 2baf e 49. Catalase 1qqw 50 r P . 47. Leucine Aminopeptidase 1lap 61. Enolase 5enGl L smd 2 cul in a2 g 1 9. Factor X 1xka, 1iod 11 k m 40 a 64. Myosin 1m8q 96 sma ptc nsporti f G 2. Phospholip Dig . ses :1 e e s a 2 tida1 a i 66 3 dg so pfk 48 llul 70. Glutaminyl-tRNA Synthetase1euq 86. DNA Photolyase 1tez Reaction ase 1poe 6. Trypsin 2ptc 43. Glutamine Synthetase 2gls 4 e 1 sla ba 7nts cul 5 C a ep 0 45 o 48. beta-Lactamase 4blm 62. Pyru5v8a.tGe lKycinearasled e1hatey3rdwe-3-phoas phaitpe ea Png sin eSpttrorarsga e: c, o2nng taeinaming nu9 trients 0. Thrombin 1ppb 12. Serum Albumin 1e7i 5 e e 1 se I e 4 55 ypi 6 50. Thymidylate Synthase 2tsc ace l od 1 s s 2 C 65. Microtubule1tub lme i j 1 2 1 14 o Center 1prc Wa nto smyal yo uc 5. y 1p xy6p0 se 5y e in rhv 2 2 11 na coCelnlsa build mea 2ny complex molecular machines that perform the biological jobs needed for life. Some of th8e8se machines Energy Production: 89 71. Isoleucyl-tRNA Synthetase 1ffy 87. Topoisomerase 1a36 - 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 44. Alcohol Dehydrogenase 2ohx Dehydrogeniase 3gpd h 13n ndi Tr, o m1 4 7 Infrastructure: 4 Protein Synthesisu: bukilding s DNA: storing and reading 51. Tryptophan Synthase 1wsy Intr d 49. Catalase 1qqw Am p o nt mol b ins: ea ur s: m s 1 2 mol ki gl a 43 71 B 51 40 snz b z1 fe . r cflor futu 1re c unountrsiu3m6u ption xo 6 o er te 66. Collagen 1bkv s . o i e l 6xka a b r 2 cto a Y 1 h E r lood P C u nj rin vi 9 e h ufe m h 9 26 72. Phenylalanyl-tRNA Synthetase 1eiy 88. RNA Polymerase 2e2i e 66 David S. Goodsell 4. Lysozyme 1lz1 8. Ribonuclease 5rsa 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate KPinase yB3pgk ppb n 25 65 H p i are molechuolxar scissors 7that cut food into digestible pieces. Others t4h6en use these piec- es to build new molecules when e x e 1 o b nAgl o 0 fr o p 94 5 52. Aspartate Carbamoyltransferase 4at1 powering the processes l 45 rote i oodstre 1 the s s 5 6 3 50. Thymidylate Synthase 2tsc . o r X 1 . 1 b F59 in n 38supporting and new mole.culaor maochindes s genetic information 52 (far left) l 2 e 7 i 38. Ferritinm 1hrs 3 h l e 1 o k 5 n h i 0 h 47 . ym i R nst i . u in 5 a s h 8 89. lac Repressor 1lbh 1efa 85 a nd de P aging te I C Molecular Machinery: D nutzri cto b i 1 bl 1 s and Antibodies: eng ng P p a pg 5 0 73. Aspartyl-tRNA Synthetase 1asy Molecular Machine60. Phrospoyglycer:ate Mutalse 3pgm R Viruse s a 95 46. Nitrogenase 1n2c stiv a j . a 1 . 3 . -p 61 . l o a 6 s of l n o h 43 51. Tryptophan Synthase 1wsy 51 e 3 oFe nm 8 g ntiboSedrie 4 ing 1 2 ni 294 se c4ells ger ow or tissues nmeed to be repaired. Some molecular machines form sturdy beaC ms that support cells, and others are of the cell ys p . 1 r moving cells 5 he k p pd e 3 D 27 L 58rdo g 63 la s m e-3 s 90. Cataabolite Gene Activator d smd 9. 2sma 3 0a 1n2sporti f a In1frastructure: Proo6i7t.e Triann sSfeyr nRNthA e4. stPnias: obui2l8do iong pfk g a pg DN8A4. :D sNtAo r1ibnnga and reading - 32 74. Ribosome 1j5e, 1jj2 a dg B

47. Leucine Aminopeptidase 1lap 61. Enolase 5enl . se 1 eTh a n the i 2 ttle in the bloodstream c5ul d3 h s yd n a 50 62 90 96 46 52. Aspartate Carbamoyltransferase 4at1 Dig 4 e 1 s. la i ba 367 m constant ba 5 5 e 3 0 t a ng Y v Blood Plasma Proteins: transporting nutrients 30 nmas (~aip esynapse)a0 odnd Atre , 2ng e acul 9 2 nd j c e . Alse 1sepmse Iodteohser s4a sth5a5t us4ey1p seien 3ergy to crawl along these6 beams. Some reco84gnize attackers and mobilize defens4e489s against infection. 30. Cytochrome c Oxidase Protein 1cgp h l1 i 5 65 68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease - rcsb.org l j e 1 e l a 42 i 75. Elongation Factor Tu/tRNA 1ttt yl c rhv 1 2 uv 6 a o te Ki ta e i d 48. beta-Lactamase 4blm 62. Pyruvate Kininastoe 1sma3aw o u 61ndi , s1 a ttl y in 37 7 supporting and Versatility!14. Rhinovirus 4rhv a new mtoe lecular 5mnacrhicnoeas n a se 2 genetic inform9a5tion 3 h 3n1 4 63. Actin 1m8q 13. Antibody 1igt 4 2 ki T u ki gen a (Complex IV) 1oco

s: m1 s 1 a . A Tour of the Protein Data Bank Am p ins: ur 60 m 2 mol . gl o 91. TATA-binding Protein/ R A Tour of the Protein Data Bank o b, o EcoRI 1eri 0 Mand deofendilng eagaincst iunjurylar Machinery: s fe u u ng 69. hThreonyl-tRNA Sy7nthetyacseer 1rqf6 er te S Molecular Machinery: b z1 xka 1 r x o l ycer 76. Elongation Factor G 1dar . o loodl P t mol p cto te Mau 28 i nj n b e 5 C h 49. Catalase 1qqw 50 1 h r g i vi 34 uv s G u yd gl m a h 944 . 74

r fe 40 P yB ppnb t ba i Al o 63 mo6v4i. nMgy ocseinl l1sm8q o H sp . fir o o hox p 7 5 96 D 31. Cytochrome c 3cyt Transcription Factor IIB 1ais

x S 66 48 . o e 1 r X 310 r1ote y 1 Fib m onogdsitnre n cutti se 2 67p. hT7r0a.n rGscfxle utrht aRemNiAn3 y4.l-ttnRaNoA S5y8notheethasldep1heuqs osResea3rwckhe5rs8 4a.r 8Do6Nu. AnD dN1b Atn hPaheo wtoloyar6s1lde 1aterez studying these molecules at the atomG ic level. These 3D structures are 9. Factor X 1xka, 1iod 11. Fibr ood h D ycer a 77. Elongation Factor Ts and Tu 1efu in 1m1j, 2baf 2 e Viruinse nst i yi h i g a s fl 5 h a s e 1 h a 8 B 45 50. Thymidylate Synthase 2tsc D zymnd decto b P i d 1. u bl R n ng g P sp Adoenoatsei nsgI le pg 5 o 52 86 o rr i s: Bi Scale: . a 73 v a a a 1 . 6 65. Microtubule1tub 3 th e 1 h o -p nl Antibody 1IGT 32. Cytochrome bc1 92. DNA Helicase 4esv . o b t sni 1 4 so of l e 4 h 72 4o 7 F m ntibSoedr iae 4 ng 1 2 68. Valyl-tRNA Synthetase 1gax e e 1 85. Restriction Endonuclease 84 P Cells buildr mcasnby c.omrplgex molecular machines that perform the biological jobs needed for life. Some of these machines Energy P3 roduction: gti 1 h bli r t arrying molecular yn se a71. Isnoleucyl-t5RNA ShynthetsasPe 1pffysepr pd s e 3 m 87. Topoisomerase 1a36 i 67 10. Thrombin 1ppb 12. Serum Albumin 1e7i ys o consta . c e s: c e a k. s freeGFPly a 1GFLvailable at the Protein Data Bank (PDB), the central storehousd e of biomolecular structures. A few 78. Prefoldin 1fxk . r 37 Hormone l m e-3 a s d 43 51. Tryptophan Synthase 1wsy 51 L 31 9 sma a 12 g a g 631. Actin 1m8q me S lo oi tei . 2P9 o 9 Troipohoosphea 5teg n a pg D An n the a 2 d d5g h h yd psfk (Complex III) 1bgy A Tour of the Protein Data Bank . Th . s: : 66. Collagen 1bkv y u o 5 d s n EcoRI 1eri Qdot525 s 93. DNA Polymerase 1tau 11 ygen cul P a S 12 4 .la 3 i f b 69.1 Tnrhmreonyl-5tRnNmA 5Syn1t0hnemtase 1qf6 l e 3 0 B 14 0 n1g nd A mcul g s i 72.c Phenylalaneyl-tRNAAle S 1ynetph(eeA .ItTaPsee)h eo1 4eisy ypise 3 88. RNA Polymerase6 2e2i e 75 79. Chaperonin GroEL/ES 1aon are molecular scissors that cut food into digestible pieces. Others t4h6en use these pieces to build new molecules when 1 od e ng ae e 5r h blood nz R nd jt P . s s s0 d s a te Ki .

52. Aspartate Carbamoyltransferase 4at1 powering the processes i e 1 ne 0 64. Myosin 1m8q mes2sages throug : uv s 6 a o 6 l a te eKixatamples from the ~100,000 structures held in the PDB are shown hel re at a mQdot800agnifica tion of about 62 o i l 33. Succinate Dehydrogenase 52 ttl r1hv t a (far left) 2 E tty Aci e/ Oax l 5 r co a En va a e 2 G 49 47 ndi , 1s a 4r 7 2 a Co s c7e0n. 2Glutatme ignyl-tRNA SynnthTetuase1e.nuqgen u s 41 86. DNA Photolyase 1tez 94. Nucleosome 1aoi 9 s thsro: ug s p 1 F s a 73. Aaspartyl-tRNA kSiynthetase k1iaosy a 89. l4a2c Repressor 1lbh 1efa 85 a 10 fe 61 e gcn ucto e . ylng es b, h moohxl o 7. gl lycer6 r yrycerer te 95 o 80. Proline cis/trans Isomerase 2cpl 29 cells grow or tissues need to be repaired. Some molecular machines form sturdy beams that support cells, and others are lood P xka t molra 65. Microtubule1tub 15. Glucagon 1gcn 9 Bi x r p e 2 x 5 o cto P te Mau n (Complex II) 1nek Viruses and Antibodies: engaging in of the cell ig g 1 vFi c mbr 3 u o o uv s s s e h G u yd 2. gl m a 3h,500,000 ti8m7.e Tso,p woisitohm eraacseh 1aat3o6m represented as a small sphere. The eno ormous range of molecular sizes is 95. HU Protein 1p51 Cells build many complex molecular machines that perform the biological jobs needed for life. Some of these machines Energy ProductBion: ppbnt ba n o e e R b lu 71. Ios1o7leucyl-et R2NAH Synthpeta.se 1ffy o o p 90. Cataabolite Gene Activator d 28 X 1 32Hormone oondgstnrthe R 1. 5 r 16 e 2 d1 1h7n4me.t aR (i nbthaoansesome. td e1hrjf)5 e=s, 1jj82ofr ehd6 h s s w 61 2 89 d 81. Heat Shock Protein Hsp90 2cg9 50 rVirus 1e y 1ssa go yui i ood g 0 acutti F s p rcx 31 o 5 D Gllucpose ycero 3 k 44 6 96 34. NADH-Quinone Oxidoreductase 62 w ulin 2hiu th h Alexa Fluor 488 s nd de in d bhil oh d m 66. Collagen 1bkv 16. Ins 4 C a e 2 s ng fl 5e h p a s te Igl h a 8 t 49 ca n g s P o h 96. Single-stranded motors that use energy to crawl along these beams. Some recognize attackers and mobilize defenses against infection. 30. Cytochrome c Oxidase cto o 858. RNA Polymerase 2e2i e are molecular sci4s1sors that c4u2 t foodc oinntost adnigte bstaibttllee p inie ctehse. Obltohoerdss threanm use these pieces to build new48 molecules when a a b bme lu a 2rr . rR n 6tsin6 s: reen th as B7i2y.n s7Pe5-h 61.eo Engely on1lanlgaantyiol-nt4R .FNaAct oSoyrs nTtuh/eteRt aN4seAh 1hetatityo -p enlill1ues 1trpagted here, frPoromte inth 1ec gwpater molecule (H2O) with only three atoms (shown at the left) to the ribosomal t e ni p e powering the processeF s m ti ntibodnie l 4Gh bl a 1 3 f yn s a10 r mnillimctaeters hp s P p s 5 s m l (Complex I) 3m9s, 3rko 82. Proteasome 4b4t o consta G lic a1 r ne (far left) 17. Epidermal Growth Factor 1eg G fer e S Scuale: 5 a a . s e-3 pd a e 3 l

(Complex IV) 1oco . r . u as g 1 me . S ci lo in oiydtei . Pl l 9 o o e 5g n as pg DNA-binding Protein 3a5u 89. lac Repressor 1lbh 1efa a 9 An 5 s tei 9 1 d o 5 h 91. TATA-binding Protein/ 85 R 13. Antibody 1igt 14. Rhinovirus 4rhv Th 3. 1 Ins t:he m p f ro: 1 2 y 4 Lu bu m yg7e3ne.h 7Ar6osp. Eaerlotdynl-gtaRtNioAn S5Fe5ya 1ncthoer dtGase1 d1aahWrs.yaPterydlase 3 snubunits with hundreds of thousands of atoms. 64 83. Ubiquitin 1ubq i i n . er s C 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u cells grow or tissues need to be repaired. S4o4me molecular machines form sturdy beams that support cells, and others are of the cell 10 1 nd A e. id mcul eg 5 Pr nz 2. R uta ndl Dj t P tec R c as . Al sep 0 deh o as te Ki se 3 91 31. Cytochrome c 3cyt 6 e s ne : l Extracellular Proteins Membr6anle Proteins Transcription Factor IIB 1ais Intracellular Protd eins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus Researchers around the world are studying these molecules at the atomic level. These 3D structures are 32 ttl1 Ep u r 1 cross the 1to a 20 E tty A4ci G e /aOhxo 74. Rulaivbos1onn2mmles 1tjid5e,5 1n56jmj2 rio10nma En vaate Ki ta 90. Cataabolite Gene Activator S 76 s a a s 77. Elonggation Factor Ts and Tu. 1efuu s throug P rp/G F Co. a cen f2o te T 1 gen B 36. Myoglobin 1mbd Adenosine Antibody 1IGT 52 7. cn , . s 3 co bo, a ep . 6 o yr 86 35 t Scale: e cto e r ynl g es r he 1 ohx r h motors that use energy to crawl along these beams. Some recognize attackers and mobili4z7e defenses against infection. 30. 3C2y.toCcyhtroocmhreo mc Oe xbidc1a se t 1 g smol a cto :9 gettBini 4gx Al r ps se 2 p m 7 lycer P ycer te Mu P9r2o. tDeiNn A1 cHgeplicase 4esv 90 87 g 1 l 9mbrto mps and Receptors3 u o uv75s. Eslaongatoion Factor T5u/tRNA 1tytdt . P freeGFPly a 1GFLvailable at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. A few nt ba ig n e F 1 e e Channels, Pu R bo 4. lu hyd o78. Preinfoeld 2inb 1l fxk . G 2 ogt1l a 67 2 12 3 4 5e Triphosphate ng bg . r i d1 eta s dhf 6 Cy5 Streptavidin/biotin2 1STP D 37. Hemoglobin 4hhb 29 Qdot525 (Com(Cpolmexp IlVe)x 1IIoI)c 1obgy Hormone un th eRcep 0 a 4 F e 2 ghen a e 41 t8sc eh ha w 5 61 6 Hormones: carrying molecular Viruse y 1ssa go yni wood p2 r 2 ecep g 4 C cutti sD op th rcx s 5 D p ycer 3 91. T9A3T.A D-bNinAd Pinogly Pmroerteaisne/ 1tau R 33 1nm 5nm 10nm d ca hi o 5 cd R m n a. e 2 7s6. Elongatfiolans eFactor G 1dar se 4 gl 0 a Luciferase 2D1S B (ATP) o rar r nd forth a to R isn6 membranes: een 5 s Bitir yn79e .A Cgme1hngnapme r(onnainno Gmreo tE2eLrD/)E =iSg 1easootnive Enz5ymes: breaking food C examples from the ~100,000 structures held in the PDB are shown here at a mQdot800agnifica tion of about 31. 3C3y.toScuhcrcoinmaete c D 3echyytdrogenase 62b me lu na h2 G n agni r t 3t back and forth across the r t4h a eN sen 1ro 1mcta21 as p wsy Phas po enl 51e 1 Transcription Factor IIB 1ais 74 68 ti l a S Researche4r1s around them weossrladg earse t hstruoduyginhg b tlhoeosde molecules at the atomic level. These 3D str4u9ctures are consta . G ulciC a h bl otei s er acto en . G Syn fero6s. ne7 S7. ciEydlonncgta tu-i6on Factothr lTas and T9u.fe 1r efous as 94. Nucleosome 1aoi 42 r ecep g me ci l i a s e 5 n B Adenosine (Complex II) 1nek . An 15 nss: m ck a P rpen F :otei 19 . Ras Protein 5p21 yAlexa41 d u uFluor4mygLeenueh8o0-tLe. iP488er1do0linqmeq wiclilsiym /n treatnerss eIs 1ionm5tneor assPemh 2acpllal snutrient molecules 86 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is 32. Cytochrome bc1 3 I er s fe R th sr 18 . L ib ta . r se 1 a a . l 92. DNA Helicase 4esv 1 . d ba g 7 e 1 tr o 95. HU Protein 1p51 P freely available at the P1r5o. Gteluinca gDoant a1g Bcnank (PDB), the central storehouse of biomolecular structures. A few 16 i Ra 2u-mAd elincrossw the s rP ne 0 nz 42 : R lu 4 l 7D8tb .Pe Ptarte fRoasldinc 1tfex dSka th yl 60 te6 K7i 70 1nm (nanometer) = Triphosphate 44 Ep . 1o ro o a r/Gs ProteinE 3sn6 tty Aci G e/Ox o . 8l 1. Hl ena2tl sSahotick yPnroteion Hsp90 2Ecng9 va D 69 Glucose Alexa Fluor 488 3(C4o. mNApDleHx -IQII)u 1inbognye Oxidoreductase . s throu18g P crh s6 pr/tG 2 19. Beta2-Adrenergic Recepto Fa Co . 2as0 h ce8n foExatrtaacelglduyllar Pr Soteinms 1. AMmey1ml.asber a1nsuem Pdr2o2teins 755. Pe9p3si.n D 5NpAe pPolymerase 1tau Intracellular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus -6 illustrated here, from the water molecule (H2O) with only three atoms (show17n at the left) to the ribosomal 7 cn B,eta l G g r 9 . s 3 co 4 ro e 1 i ep n a 6 yr 23 96. Single-stranded 6 78B 10 millimeters (ATP) examples from the ~10160. ,I0ns0u0li ns 2trhuiuctures held in the PDB are15 show1n6 here at a magnification of about 1ge g .ls acto a 2 ceto 9 to 3 9Ex.uBi 4964 xyl Al r enm,s 7s9.. C8a2hs .a Em.sPpere 2ortoepnaishnooah Gm519xmreo Er4bLb/4EtS nm1aon . P Scale: 33. Succ(iCnoamtep Dleexh Iy)d 3romg9esn,a 3serk o n 1 1e9 F m1 , e 8 bgemt br 20. Acetylcholine Receptor 2bg9 3 R bo 4. luo hyd49 ymin e p2 bl a 2. Phosp2holti1pase 1poe 6. Try9p4s.i nN u22cplte2co4some 1aoi Hormone u n t.hAcetylderp2 jwa Recefp8 2 m2 lut 64 0. ar 4 F Di d1 gene. tTah asto e 41dhf tsc 6 a 5 DNA-binding Protein 3a5u62 25 Storage: containing nutrients 77 88 Water subunits with hundre1d7.s Eopfi dtheromuasl aGnrodws toh fF acttoomr 1se.gf 3(C5o. mATpPl eSxy nIIt)h 1ansee k1e79, 1c17, 1l2p, 2a7u ssa go hi n o2w0 i 5 2 dc R 1 r m 4 ec e3p 18 eceptor 4 C . e 2 8o0.thP80r3o. lUinbyepi qcauissi/ettinrtae 1n Cus bIsqomeras4e8 2cpel 4 0 80 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is uca 2ha r nd fEoprntho ,angi sin to n r Rel sn6 21. Epidermal Growth Factor R een 45as itr yn5ne AgmTernm srta se 2 3. De-1oaxsyribonu-1cle a5se1 2dnj 7. Ca9rb5o. HxyUp ePprotidteaisne 13pcp51a 13 71 Extracellular1 Pnrmoteins 5nmMem1b0rnamne Proteins Intracellular Proteins: Cytosol me Gl inC l G 1. tei iv ear p tei ctonn n 3 Intracelluϕlar P19roGter ins: Cyto. Nseo 8Sl1. cHi er1oa. t Shcoptacak Proteianp Hspw9s0y 2cg9 5 Intracellular Proteins: Nucleus for future consumption Glucose 34. 3N6A.DMHy-oQguloinboinne1 Omxbiddoreductase . ul a 2 ro 1 esn do ecerpo a a 1ivo, 2jwa, 2gs6 =. 0.92,cifer46 in e uεyd 5=acta Au71,000s qqw thla e 1 Msfer cm illustrated here, from the water molecule (H2O) with only three atoms (shown at the left) to the ribosomal 15 ns m ck a P pr ho p F otseui4 19 41 u m L eh -L ed. yn s 4. anLysozyme 1lz1 8. Rib9o6n. uScinlegalese-s t5rarsnaded 87 92 (Complex I) 3m9s, 3rko . I derba as m-Ad R ne R th Ch pr 1 . L ta 7. 82. Pro5t2eaes 1ome 4sbe4 1tha tr 38. Ferritin 1hrs 38 78 Cy5 12Streptavidin/biotin 3 1STP 4 5 37. Hemoglobin 4hhb 16 i R u2 2. olicroycsoosw tuhme ms P prh 22. Rhodopsin 1f88 42 lu 4ol D beatate Rlas c ated Sa yl DNA-binding Protein 3a5u 79 Channels, Pumps and Receptors: getting 33 Ep 8. P 2 P-gl r si /Gu e 1 64 . G h 8. l ta n2 yl ti yn o 49 1nm (nanometerD) =Wig aetesrtive Enszuybmuneitss: wbirthe ahkuinndgrLuciferase efodso dof t h2D1Sousands of atoms. 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u 7. 1 B, eta ch. l Gtas gs365 rP as r 9 Brightness3 co 48o3fo. CUabei q1~uiitd in65e p1unb Sq am 1 9l.s 23 a o , 2 mto 9 to 3 23. P-glycoprotein 4m2t 445 Al r . as ymop ha m rb 68 76 Photostability! -6 back and forth across the membrane e1 m. P u 8 t . yd 49 in p bl a 2 91 10 millimeters 36. Myoglobin 1mbd n . Acetylder 41jwa ealcceipf8 xbyggme2n lut 44 ih gen . Th toe 4 tsBc lood Palt1asma Proteins: tran5sporting nutrients into small nutrient molecules 21 n 20 i 5p22 2 C 1 or o2 4 ece 3p s: 18 24. Potassium Channel 3lut . D o 0 ypas te C 48 0 87 18. Ras Protein 5p21 a nd foEprntho a, g5ic. Rsin to n r R el si (Cy5: 4~68546 itr 5@e A mT646/664rm rta se 2 7nm)0 se 4 5 12 3 4 5 37. Hemoglobin 4hhb h 1. tei iv er2 p Cyctlei nn e p 1su4 . N cin 61.9 pa a 47 wsy a Extracellular Proteins 1. AMmeymlasber a1nsem Pdroteins 5. Pepsin 5pep Intracellular Proteins: Cytosol 33 C 2 o 1 do ec.epro actoa h 25. Calcium Pum Intracellular Pro6teins: Cyt5oascotal As th andsfe rdefending against injury Intracellular Proteins: Nucleus 34 Digestive Enzymes: breaking food 19. Beta2-Adrenergic Receptor/Gs Protein6 3sn6 78 ck a Pr ren o 26 p F sut4 4 Leu -L . qqw yn se 1 n 20 ba s Rh e R th Ch p y1n ooxygenase 1prh 7. 52e 1 a tra 68 26 2. Phospholipase 1poe 6. Trypsin 2ptc 22 Storage: containing nutrients Ra 2-Ad2. lin ycow um ms pr h 26. Cycl 4 beta as te S th yl9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf 73 20. Acetylcholine Receptor 2bg9 23 8. 2 o P-gl ro si uto e 1g . tal yla yn o 49 88 72 into small nutrient molecules 1 Beta ch . l Gtas s6 Po as in 9 48 a id n S am 10. Thrombin 1ppb 12. Serum Albumin 1Qdote7i 525 streptavidin 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 24 . 23 a o 2g mh st m 3 HaloTag18 4 5 . C a rb 70 71 54 21. Epidermal Growth Factor Receptor for future consumption 25 19 m . P , ciuP8 ygenet o 969 ym ph a 11 12 1. A4m. yLlyassoez 1ymsme d1lz1 5. P8e.pRsiinb o5npuecplease 5rsa . Acetyl er24 wa al f8 x rv ut fr 4 Th to 14 1ivo, 2jwa, 2gs6 6 1978 0 id j 3C8 1 oo am2 3ly s: 8 0. yp te C 8 38. Ferritin 1hrs 2 Ep o, 2 5. sin nh 4 elrg si II 1 Photosynthesis: 46 5 Tr rta 4 Ex.77 488 nm (broad),9 Em. 525 nm53 82 2. Phospholipase 1poe 6. Trypsin 2ptc Storage: containing nutrients 1. iv 2 p Cytceli ne e 43 SNAP-tag 1. pa 47 10 81 22. Rhodopsin 1f88 2 1 do 6. ro aenn thun tem 5 As Viruses and Antibodies: engaging in 88 93 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 ho 2 p Ch 1snu4 s sys g harvesting 39 2. 71 80 89 23. P-glycoprotein 4m2t for future consumption . R yco m psye pr h to tin CLIP-tag 5 ϕ -1 -1 4. LBysloozyomde P1llza1sma Prot8e. iRnibso:nturcalenasep 5orsrating nutrients 22 -gl siu umo the 1 g ho t constant battle9 in the bloodst r=ea m0.30, ε = 130,000 M cm Qdot525 . P s Pot as in P l rves w energy from 4 ng a7n8d joining the molecules of life 24. Potassium Channel 3lut 38. Ferritin 1hrs 23 38ota mh st 7. sm5 a r 5 tosolEnzymes: cutti 92 and defending against injury . P uP e 2 ro1 t-h x 1 c 4 13. Antibody 1igt y 14. Rhinovirus 4rhv 79 55 34 4 alci xygrevn f igh ple eti the sun dgk 25. Calcium Pump 1su4 2 C oo a y s. :L m th ins: C 39. FattBrightnessy Acid Synthase 2uvb, 2uv c~ 395 3@. He x488okinase 1 nm 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf 5. h rg s8i IIsyn 40 3 46 Blood Plasma Proteins: transportin2g6 .nCuyctlroioexnygtesnase 1prh 2 Cycl e e2 Co to n II 4 72 47 rote 73 54. Phosphoglucose Isomerase 1hox 90 6. n hun tehmo o 27. Photosystem r P 40. RuBisCo: Ribulose Bisphosphate 56 10. Thrombin 1ppb 12. Serum Albumin 1e7i 2 e nt s ysP cti g prc a 83 11 26 y e tos9. ea n 1 7 1s5l Hormonelslu:l carrying moleCcaurBrightnessbloaxrylase/Oxygenase 1rcx ~ 975 5@. Ph o405sphofructo nmkinase 4 pfk 93 and defending against injury 18 12 14 sth o 2 R ttier 2 ce 34 to gPh l ves t g Live cell anda 74 o tin7. arCenrw 28. Light-harvestin Intr 41. Green Fluorescent Protein 1gfl 56. Aldolase 4ald 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2bafPhotosyn9thesis: h s 2 1ms5 t-h 5 14 c messages through bytolosool d 10 P e ro lex eti Complex 1rwt 72 73 hate Isomerase 2ypi 57 Viruses and Antibodies: engaging in rv f Ligh p th 4 (Qdot42. Luciferase 800:2d1s ~90089 @ 532/80057. Triosephosp nm 10. Thrombin 1ppb 12. Serum Albumin 1e7i 53 a y . m I yn 0 4 15. Glucagon 1gcn ins: C harvesting 11 12 h rg 28 Co I s 249. Photosynthetic 3 thetase 2gls 58. Glyceraldehyde-3-phosphate 75 constant battle in the bloodstream 39 14 e n oto n 4 16. Insulin 2hiu rote 43. Glutamine Syn15 16 17 en u temh o prc Reaction ar P se 3gpd 94 energy9 from s sys. P cti g 1 2 in vivo imaging!ul 81 44. Alcohol andD82ehydro g~4600enase 2ohx @ 405/800Dehydrogen anm) e 9 ea n 7 4 17. Epidermall lGrowth Factor 1egf 13. Antibody 1igt 14. Rhinovirus 4rhv 10 th oto2 R titer 2 Center 1prc ce ate Kinase 3pgk 59 Viruses and Antibodies: engaging in Enzymes: cutting and joining the molecules of life Ph l ves t a 45. Dihydrofolate Redu8c9tase 1dhf 59. Phosphoglycer the sun 7. ar Cenrw Intr 93 2 1s5 t-h 1 c 8 ytosol 60. Phospoglycer9a0te Mutase 3pgm constant battle in the bloodstream 55 lex eti 2 1 46. Nitrogenase 1n2c 27. Photosystem II 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1dgk Ligh p th 6 4 44 58 Hormones: carrying molecular . m yn 6 0 Channels, Pumpins: Cand Receptors: getting ase 1lap 61. Enolase 5enl 13. Antibody 1igt 14. Rhinovirus 4rhv 40. RuBisCo: Ribulose Bisphosphate 54. Phosphoglucose Isomera 28 Co s 4 27 47. Leucine Aminopeptid 35 1s5l se 1hox oto n Green Fluorescent Protein rote 74 w 76 h o 56prc back and foar Pth across the metea-mLabctarmanasee 4blm 62. Pyruvate Kinase 1a3 60 91 messages through blood 28. Light-harvesting Carboxylase/Oxygenase 1rcx 55. Phosphofructokinase 4pfk . P cti 1 42 ul 48. b 29 Rea ter 27 ll 21 90 15. Glucagon 1gcn Complex 1rwt 41. Green Fluorescent Protein 1gfl 56. Aldolase 4ald 18. Ras Proacte in 5p21 49. Catalase 1qqw Hormones: carrying molecular Cen 4Ex.0 488 nm, Em.75 507 nm Intr 83 16. Insulin 2hiu 29.1P5hotosyn16thetic 17 42. Luciferase 2d1s 57 28 19. Beta2-Adre4n5ergic Re74ceptor/G5s0 P. rTohtyemini d3yslna6te Synthase 2tsc 93 57. Triosephosphate Isomerase 2ypi 6 66 41 4 20 22 messages through blood Reaction 6 29 4 20. A-1cetylchol-1ine Receptor 2bg951. Tryptophan Synthase 1wsy 51 23 95 17. Epidermal Growth Factor 1egf 43. Glutamine Synthetase 2gls 58. Glyceraldehyde-3-phosphate C ϕ = 0.60, ε = 56,00043 M cm 15. Glucagon 1gcn Center 1prc C 46 52. Aspartate Carbamoyltransferase 4at1 24 96 Infrastructure: Protein Synthesis: building 44. ADlcNohAol: Dsethoyrdirnoge naansed 2 roehaxding Dehydrogenase 3gpd - 21. Epidermal Growth Factor Receptor 25 36 B 42 75 61 16. Insulin 2hiu 15 16 17 65 Y 1ivo, 2jwa, 2gs6 19 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate Kinase 3pgk- EGFP Brightness ~ 34 77 Channels, Pumpssu papnodr tRinegce apntdo rs: getting new molecular machines genetic information 3 59 m 50 94

. n

M 0 olecular 22. Rhodopsin 1f88 Machinery: 17. Epidermal Growth Factor 1egf Molecular Machinery: 0 46. Nitrogenase 1n2c 35 60. Phospoglycerate Mutase 3pgm 8 1 28 48 80 2 76 30 63 moving cells 27 D 58 1 91 back and forth across the membrane 67. Transfer RNA 4tna 47. L8e4u. cDinNeA A 1mbinaopeptidase 1lap 61. Enolase 5enl 66 9 4 ins 23. P-glycoprotein 4m2t a 2 (mNeonGreen: ~93 @ 506/517 nm) 10 nm 21 10: nmnm rote rcsb.org 68. Valyl-tRNA Synthetase 1gax 85. Restriction Endonuclease 84 v le 5 P 78 92 18. Ras Protein 5p21 48. beta i a 24. Potassium Channel 3lut 37 -Lactamase 4blm 62. Pyruvate Kinase 1a3w d A Tour of the Protein Data Bank 31 Channels, Pumps a63n. dAc Rtine1cme8pqtors: getting Sc ) = ane 79 A Tour of the Protein Data Bank EcoRI 1eri 60 52 19. Beta2-Adrenergic Receptor/Gs Protein 3sn6 69. Threonyl-tRNA Synthetase 1qf6 49. Catalase 1qqw 35 S m eter 25. Calcium Pump 1su4 40 . n mbr 76 47 64. Myosin 1m8q 20 22 1 m 91 back and forth across the membrane 86. DNA Photolyase 1tez G o s Me 20. Acetylcholine Receptor 2bg9 66 70. Gluta2m3inyl-tRNA Synthetase1euq 45 50. Thymidylate Synthase 2tsc an nm 26. Cyclooxygenase 1prh 65. Microtubule1tub 21 o e1t0er 29 62

o (n Cells build many complex molecular machines that perform the biological jobs needed for life. Some of these machines Energy Production: 18. Ras Protein 5p21 71. Isoleucyl-tRNA Synthe2t4ase 1ffy 87. Topoisomerase 1a36 m lim 49 26 21. Epidermal Growth Factor Receptor d l ns 43 2551. Tryptophan Synthase 1wsy 51 9 1n i ins i 18 66. Collagen 1bkv 88. RNA Polymerase 2e2i s 2 -6 mm 41 19 e : n are molecular scissors that cut food into digestible pieces. Others then use these pieces to build new molecules when powering the processes 19. Beta12iv-Aod, r2ejnwearg, i2cg Rse6ceptor/Gs Protein 3sn6 72. Phenylalanyl-tRNA Synthetase 1eiy 46 52. Aspartate Carbamoyltransferase 4at1 le 05 rote Prote 42 20 22 l a 1 77 54 (far left) l c r P ne 23 89. lac Repressor 1lbh 1efa S a ) = a Photosynthesis: 20. Acetylcholine Receptor 2bg9 85 a cells grow or tissues need to be repaired. Some molecul 22. Rhodopsin 1f88 73. Aspartyl-tRNA Synthetase 1asy 61 llul 44 95 ar machines form sturdy beams that support cells, and others n 80 are of the cell 24 nm ce eter mbr 53 32 21. E2p3i.dPe-rgmlyacol Gprrotweitnh 4Fmac2totr Receptor 28 74. Ribosome 1j5e, 1jj2 25 90. Cataabolite Gene Activator 50 d 1 a om s Me harvesting xtr n m 96 motors that use energy to crawl along these beams. Some recognize attackers and mobilize defenses against infection. 30. Cytochrome c Oxidase t E a n 39 124iv.oP,o 2tajwssaiu, m2g sC6hannel 3lut 19 75. Elongation Factor Tu/tRNA 1ttt Protein 1cgp 48 h Sc a(nle: 1e0ter 78 92 81 82

e m (Complex IV) 1oco m illi ns 77 energy from 79 n ins i

91. TATA-binding Protein/ R 1 22. R25h.oCdaolpcisuinm 1 Pf8u8mp 1su4 76. Elongation Factor G 1dar 1n-6 mm 5nm 10nm 80 Enzymes: cutting and joining the molecules of life C : n 93 Researchers around the world are studying these molecules at the atomic level. These 3D structures are 31. Cytochrome c 3cyt Transcription Factor IIB 1ais le 105 rote Prote the sun 23. P2-6g.lCycyocploroxteyigne n4amse2 t1prh 77. Elongation Factor Ts and Tu 1efu S ca ar P ne 55 Adenosine B ) = 32. Cytochrome bc1 47 86 52 S lul a 78 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1dgk 92. DNA Helicase 4esv l 27. Photosystem II 92

P m eter Triphosphate freely available at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. A few 24. Potassium Channel 3lut 29 78. Prefoldin 1fxk 67 26 n ace m mbr

D 1 79 eus (Complex III) 1bgy 18 93. DNA Polymerase 1tau x1trnmn (onanosmeter) =M e 1s5l 40. RuBisCo: Ribulose Bisphosphate 54. Phosphoglucose Isomerase 1hox Intracellular Proteins: Nucl (ATP) 25. Calcium Pump 1su4 B E a Intracellular Proteins: Cytosol examples from the ~100,000 structures held in the PDB are shown here at a magnification of about 33. Succinate Dehydrogenase 79. Chaperonin GroEL/ES 1aon 62 1 0(n-6 milleimtereters Carboxylase/Oxygenase 1rcx 56 Photosynthesis: 41 42 94. Nucleosome 1aoi 49 54 m llim 28. Light-harvesting 55. Phosphofructokinase 4pfk 26. Cyclooxygenase 1prh 80. Proline cis/trans Isomerase 2cpl 1n i ins 3,500,000 times, with each atom represented as a small sphere. The enormous range of molecular sizes is (Complex II) 1nek -6 m Complex 1rwt Intracellular Proteins: Cytosol 41. Green Fluorescent Protein 1gfl 95. HU Protein 21563p51 ac0ellular Prrooteins Membrane Proteins 56. Aldolase 4ald 83 Glucose 34. NADH-Qu harvesting 81. Heat Sh4o4ck Protein Hsp90 2cg9 Extr 1 r P 93 illustrated here, from the water molecule (H2O) with only three atoms (shown at the left) to the ribosomal inone Oxidoreductase 18 39 96. Single-stranded a 29. Photosynthetic 42. Luciferase 2d1s 57. Triosephosphate Isomerase 2ypi 57 (Complex I) 3m9s, 3rko 82. Proteasome 4b4t llul 81 82 Pheontoesrgyyn tfhroemsis : Scale: DNA-binding Protein 3a5u 54 ace Reaction 43. Glutamine Synthetase 2gls 58. Glyceraldehyde-3-phosphate Water subunits with hundreds of thousands of atoms. 64 83. Ubiquitin 1ubq xtr 35. ATP Synthase 1e79, 1c17, 1l2p, 2a7u the sun 1nm 5nm 10nm Enzymes: cutting and joining the molecules o5f3 life E Center 1prc 93 44. Alcohol Dehydrogenase 2ohx Dehydrogenase 3gpd harvesting 55 36. Myoglobin 1mbd 39 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1dgk 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate Kinase 3pgk 94 12 3 4 27. Photosystem II 87 81 82 59 5 37. Hemoglobin 4hhb energy from 46. Nitrogenase 1n2c 60. Phospoglycerate Mutase 3pgm Digestive Enzymes: breaking food 33 1s5l 1nm (nanometer) = Enz40y.mRueBiss:Coc:u Rtibtiunlogs ea Bnisdp hjosipnhianteg the m54o.lPehcousplehosg olufc olsifee Is omerase 1hox the sun Carboxylase/Oxygenase 618rcx 56 27 93 47. Leucine Aminopeptidase 1lap 61. Enolase 5enl 58 28. Light-harvesting 10-6 millimeters 55. Phosphofructokinase 4pfk 55 into small nutrient molecules 27. PhoCtoosmysptelemx 1IIr wt 39. F41a.ttyG Areceidn SFylunothreascee 2nut vPbro, t2euinv c1gfl 53. H56e.xAolkdinoalassee 1 4dagldk 83 48. beta-Lactamase 4blm 62. Pyruvate Kinase 1a3w 69 70 60 1. Amylase 1smd 5. Pepsin 5pep 6 78 129s5. lPhotosynthetic Extracellular Proteins Membrane Proteins 40. R42u.BLisuCcoif:e Rraibseu l2odse1 sBisphosphate 5In4t.raP5c7he.ollTsurpiloahrso ePgprluohctoeosisnpesh :Ia sCtoeym tIosesoromalseer a1sheo 2xypi 57 Intracellular Proteins: Cytosol 93 Intra4ce9l.luClarta Plarsoete 1inqsq: wNucleus 56 40 2. Phospholipase 1poe 6. Trypsin 2ptc Storage: containing nutrients 28. LighRt-ehaacrtvioenst ing C43a.rbGoluxytalamsein/Oe xSyygnethneatsaes e1 r2cgxls 55. Phosphofructokinase 4pfk 66 45 50. Thymidylate Synthase 2tsc 58. Glyceraldehyde-3-phosphate 88 3. Deoxyribonuclease 2dnj 7. Carboxypeptidase 3cpa 13 for future consumption ComCpelnetxe r1 r1wptrc 41. G44re. eAnlc oFhluool rDesecheyndt rPorgoetneians e1 g2folhx 7516. AldoDlaeshey d4raoldgenase 3gpd 83 43 51. Tryptophan Synthase 1wsy 51 4. Lysozyme 1lz1 8. Ribonuclease 5rsa 29. Photosynthetic 42. L4u5c. iDfeirhaysder o2fdo1laste Reductase 1dhf 57. T5r9io. sPehpohsopshpohgalytece Irsaotme eKrinasaes e2 3ypigk 57 27 4963 28 38. Ferritin 1hrs 38 59 94 52. Aspartate Carbamoyltransferase 4at1 Reaction 43. G46lu. tNamitroingee nSyanseth 1enta2sce 2gls 58. G60ly.cPehroalsdpeohgyldycee-3ra-pteh Mosupthaasete 3 pgm 61 95 Center 1prc 27 44. A47lc.oLheoulc Dineeh Aydmroingoepneapsteid 2aoseh x1lap D61e.hEyndorolagseen 5aesen l3gpd 58 28 50 96 Blood Plasma Proteins: transporting nutrients 48 45. D48ih. ybdertoa-foLalacttea mReadseu c4tabslme 1dhf 59. P6h2.oPspyrhuovgaltyec eKriantaes eK i1na3sew 3pgk 94 and defending against injury Semi-genetically encoded fluorescent proteins59 60 Outline 46. Nitrogenase 1n2c 60. Phospoglycerate Mutase 3pgm 34 27 40 49. Catalase 1qqw 58 9. Factor X 1xka, 1iod 11. Fibrin 1m1j, 2baf 66 45 47. Leucine Aminopeptidase 1lap 61. Enolase 5enl 72 50. Thymidylate Synthase 2tsc 73 47 52 10. Thrombin 1ppb 12. Serum Albumin 1e7i 48. beta-Lactamase 4blm 62. Pyruvate Kinase 1a3w 29 11 12 43 51. Tryptophan Synthase 1wsy 51 60 14 40 46 49. C52a.taAlaspsea r1taqtqe wCarbamoyltransferase 4at1 62 9 66 41 42 49 Viruses and Antibodies: engaging in 10 45 50. Thymidylate Synthase 2tsc 61 95 HaloTag,28 SNAP-tag,43 and CLIP-tag are51. Trypto pexampleshan Synthase 1wsy of 50genetically51 89 encoded proteins that 44 96 constant battle in the bloodstream 48 46 52. Aspartate Carbamoyltransferase 4at1 Scale: 13. Antibody 1igt 14. Rhinovirus 4rhv 61 95 form covalent bonds with synthetic membrane-permeable fluorophores. 1nm 5nm 10nm 28 50 96 47 48 52 90 Hormones: c 29 arrying molecular 1nm (nanometer) = 49 74 62 messages through blood 41 42 10-6 millimeters 47 52 15. Glucagon 1gcn 29 44 16. Insulin 2hiu 15 16 17 75 Extracellular Proteins Membrane Proteins Intracellular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus Scale: 49 62 17. Epidermal Growth Factor 1egf 41 42 1nm 5nm 10nm HaloTag 44 Channels, Pumps and Receptors: getting Scale: Fluorescent proteins (FPs) 35 1nm (nanometer) = 76 back and forth across the membrane 1nm -6 5nm 10nm 91 21 10 millimeters 18. Ras Protein 5p21 19. Beta2-Adrenergic Receptor/Gs Protein 3sn6 Ex1trnamce l(lunlaanr oPmroetteeinr)s = Membrane Proteins Intracellular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus 20 22 20. Acetylcholine Receptor 2bg9 23 10-6 millimeters 21. Epidermal Growth Factor Receptor 24 Other fluorophore technologies 25 1ivo, 2jwa, 2gs6 19 Extracellular Proteins Membrane Proteins Intracellular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus 77 22. Rhodopsin 1f88 80 23. P-glycoprotein 4m2t 24. Potassium Channel 3lut 78 92 79 25. Calcium Pump 1su4 Single FP-based biosensors 26. Cyclooxygenase 1prh 26 18 Protein of Interest Protein of Interest Photosynthesis: 54 harvesting 53 39 energy from 81 82 the sun Enzymes: cutting and joining the molecules of life 93 55 27. Photosystem II 39. Fatty Acid Synthase 2uvb, 2uvc 53. Hexokinase 1dgk - Halotag is a mutant of a bacterial haloalkane dehydrogenase 1s5l 40. RuBisCo: Ribulose Bisphosphate 54. Phosphoglucose Isomerase 1hox 28. Light-harvesting Carboxylase/Oxygenase 1rcx 55. Phosphofructokinase 4pfk 56 Complex 1rwt 41. Green Fluorescent Protein 1gfl 56. Aldolase 4ald - SNAP- and CLIP-tag are engineered variants of the83 DNA repair protein O6- 29. Photosynthetic 42. Luciferase 2d1s 57. Triosephosphate Isomerase 2ypi 57 93 Reaction 43. Glutamine Synthetase 2gls 58. Glyceraldehyde-3-phosphate Center 1prc 44. Alcohol Dehydrogenase 2ohx Dehydrogenase 3gpd alkylguanine-DNA alkyltransferase 45. Dihydrofolate Reductase 1dhf 59. Phosphoglycerate Kinase 3pgk 59 94 46. Nitrogenase 1n2c 60. Phospoglycerate Mutase 3pgm 27 47. Leucine Aminopeptidase 1lap 61. Enolase 5enl 58 Keppler et al. Nat. Biotechnol., 2003, 21, 86 (SNAP-tag); Gautier et al. 2008, 15, 128 (CLIP-tag); Los et al. ACS Chem. Biol., 2008, 3, 373 (HaloTag). 48. beta-Lactamase 4blm 62. Pyruvate Kinase 1a3w 60 40 49. Catalase 1qqw 66 45 50. Thymidylate Synthase 2tsc 43 51. Tryptophan Synthase 1wsy 51 46 52. Aspartate Carbamoyltransferase 4at1 61 95 28 50 48 96

47 52 29 62 41 42 49 44 Scale: 1nm 5nm 10nm

1nm (nanometer) = 10-6 millimeters

Extracellular Proteins Membrane Proteins Intracellular Proteins: Cytosol Intracellular Proteins: Cytosol Intracellular Proteins: Nucleus Figures

Figure 1. Design and in vitro characterization of KIRIN1 (a) Design and construction ​ ​ ​ of the genetically encoded K+ indicator KIRIN1. mCerulean3 (residues 1-228) at the ​ N-terminus is linked with Kbp (residues 2-149), followed by cp173Venus (full length) at the C-terminus. (b) Schematic demonstration of the molecular sensing mechanism of ​ ​ KIRIN1. Binding of K+ induces a structural change in the Kbp protein, increasing the ​ FRET efficiency between mCerulean3 and cp173Venus in KIRIN1. (c) KIRIN1 emission ​ ​ fluorescence spectrum (excitation 410 nm, emission 430 nm to 650 nm) in Tris HCl

+ buffer with (red) and without (blue) K .​ (d) FRET acceptor-to-donor fluorescence ratio ​ ​ ​

+ (F530/F475) of purified KIRIN1 protein at various concentrations (1 µM to 150 mM) of K ​ ​ ​ ​ ​ (red) and Na+ (blue) solutions. (e) FRET acceptor-to-donor fluorescence ratio of purified ​ ​ ​ 29 30 KIRIN1 protein upon addition of various physiologically relevant ions including Mg2+ (10 Designs of fluorescent protein-based biosensors​ Roger Tsien’s Ca2+ biosensor equation Biochemistry: Baird et al. Proc. Natl. Acad. Sci. USA 96 (1999) 11245 2+ 2+ mM), Ca ​ (10 µM), and Zn (10​ µM). 2+ 2+ ​ ​ Förster resonance energy transfer Fluorescent protein reconstitution fluorophore + Ca binding motif = Ca biosensor O OH HO O

HO OH N N O O O O O O O O O Fura-2 N N N HO OH O O O OH HO O N HO O O BAPTA

Split FPs HO O

A Y B A B

Single fluorescent protein FIG.5. FluorescenceofatypicalHeLacellexpressingcytosolic EYFP–calmodulin insert as a function of time after successive addi- Calmodulin tions of reagents. The fluorescence normalized by the initial fluores- Dimerization-dependent FPs cence F0 is plotted on a logarithmic scale. + (CaM) = affinity (apparent Kd Ϸ 0.4 mM) were modest, they indicate that the sensitivity of EYFP fluorescence to the conformation >1000 works cited! Review of an inserted receptor may be of some generality. Cite This: Chem. Rev. XXXX, XXX, XXX−XXX pubs.acs.org/CR

Genetically Encoded Fluorescent Biosensors Illuminate the DISCUSSION Spatiotemporal Regulation of Signaling Networks Despite the gross rearrangements of their primary sequence Eric C. Greenwald,† Sohum Mehta,*,† and Jin Zhang*,† Camgaroo (1999) described above, GFPs can still fold correctly, generate inter- †University of California, San Diego, 9500 Gilman Drive, BRFII, La Jolla, CA 92093-0702, United States nal chromophores, and protect their excited states from vi- Miyawaki et al., Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2135; Ghosh et al., J. Am. Chem. Soc. 2000, 122, 5658; Kerppola, Chem. Soc. Rev.*S Supporting 2009 Information, 38, 2876; Nagai et al., Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3197; Nakai et al., Nat. brational deactivation. Circular permutation is known (5) to be tolerated in a variety of proteins when the original N and C Biotechnol. 2001ABSTRACT:, 19,Cellular 137; signaling Alford networks et al., are theChem. foundation Biol. which 2012 determines, the19, fate 353; Ding et al., Nat Methods 2015, 12, 195. Grynkiewicz, Poenie, and Tsien, J Biol Chem. 1985, 260, 3440; Baird et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11241. and function of cells as they respond to various cues and stimuli. The discovery of termini are fairly close in space, as is true for GFPs. If a protein fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the consists of more than one autonomous domain loosely held spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we together by flexible linkers, it is easy to imagine that its 22 endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we function could be preserved despite swapping the order of review how the high temporal and spatial resolution afforded by fluorescent biosensors those domains. However, GFPs would seem poor candidates has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research because of their monolithic cylindrical symmetry, intricate in both biosensor design and applications that are on the forefront of biosensor development. interdigitation of the 11 strands making up their ␤-barrels, complexity of their maturation process, and intolerance of CONTENTS 3.4. How Fast Can We Go? AO 4. Obtaining Spatial Information from Biosensors AP significant N- and C-terminal truncations (21). Nevertheless, 1. Introduction B 4.1. Membrane-Associated Signaling Compart- an unbiased genetic screen located at least 10 different sites 2. Designing Genetically Encoded Biosensors B ments AR 2.1. Elements of Genetically Encoded Biosensors B 4.1.1. Partitioning by Membranes AR (Table 1 and Fig. 2) within the EGFP sequence at which a 2.2. Biosensors for Monitoring Protein Behavior M 4.1.2. Partitioning within Membranes AT 2.2.1. Protein Expression, Turnover, And Lo- 4.2. Compartmentation by the Activity of Path- permuted fluorescent protein could be initiated after a con- calization M way Regulators AT 2.2.2. Protein−Protein Interactions M 4.2.1. Calcium Signaling AT stant N-terminal purification tag. Currently, we have no ex- 2.3. Biosensors for Monitoring Biochemical 4.2.2. cAMP Signaling AV planation for the locations or varying phenotypes of the Activity Dynamics N 4.2.3. Other Signaling Molecules AX 2.3.1. Inducing the Translocation of a Fluo- 4.3. Assembly of Macromolecular Signaling permissible permutations, but they should be interesting test rescent Protein N Complexes AY 2.3.2. Directly Sensitizing the Chromophore Downloaded via UNIV OF ALBERTA on December 20, 2018 at 04:55:15 (UTC). 4.3.1. Scaffolding by A-Kinase Anchoring cases for a future understanding of protein folding. Also, we do of a Fluorescent Protein P Proteins AY 2.3.3. Engineered Modulation of the Photo- 4.3.2. Molecular Scaffolds in Other Signaling not yet know whether the increased acid sensitivity of the See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. physical Behavior of a Fluorescent Pathways AZ fluorescencewasduetothecircularpermutationsthemselves, Protein R 5. Computational Models and Fluorescent Biosen- 2.3.4. Coupled Reporter Systems AG sors AZ the attachment of the constant N-terminal polyhistidine tag, or 2.4. New Biosensor Classes AG 5.1. Computational Modeling Background AZ 31 32 2.4.1. Sensors Based on Infrared Fluorescent 5.2. Examples of the Integrated Approach BB to the nature of the linker joining the former N and C termini. Proteins AG 5.2.1. Analysis of Temporal Dynamics BB 2.4.2. Biochemical Activity Integrators As in 5.2.2. Evaluation of Spatial Compartmentali- Fig. 6 summarizes the many topologies for merging the Vivo Snapshot Reporters AH zation BB 2+ 2.4.3. Fluctuation-Based Biosensors AI sequences of a host protein and a GFP or cpGFP. cpGFPs 5.2.3. Analyses of Cellular Heterogeneity BD The growing toolbox of (non-Ca ) single GFP-based biosensors GCaMP: the prototypical Ca2+ biosensor (2000)3. Obtaining Temporal Information from Biosen- 5.2.4. Information Theory BD represent a library of mutants whose spatial orientations with sors AJ 6. Pushing the Field Forward BE 3.1. Kinetics of Individual Reactions AL 6.1. Improved Resolution BE respect to fusion partners should differ from those of regular 3.2. Higher-Order Signaling Dynamics AM 6.2. Multiplexed Biosensor Imaging BF 3.2.1. Adaptive Responses AM GFP mutants. Thus, when conventional tandem fusions to 3.2.2. Oscillations AN 3.2.3. Bistability and Ultrasensitivity AN GFPs are unsatisfactory either in the function of the host Received: May 25, 2018 Loren Looger (LL) Lin Tian (LT) Protein Kinases (JZ) 3.3. Importance of Single-Cell Readouts AO protein or in FRET to or from the GFP, a tandem fusion to IG © XXXX American Chemical Society A DOI: 10.1021/acs.chemrev.8b00333 F .4. In vitro characterization of EYFP–calmodulin insert protein. acpGFP(Fig.6f)mightbeworthtrying.ThehigherpKavalues Chem. Rev. XXXX, XXX, XXX−XXX (A)AbsorbancespectrawithEDTAandzeroCaExRai-AKAR2 ϩ,orwithsaturating of cpGFP mutants might also be exploited to measure the pH 2+ Ca2ϩ,bothatpH7.5andthesameproteinconcentration.(B)Fluores- Ca in relatively alkaline compartments such as mitochondria. Like cence excitation andExRai-CKAR emission spectra obtained under the same condi- the pHluorins (15) and a recent nonpermuted GFP mutant, tions. (C)TitrationatpH7.5withCa2ϩ buffers, plotting fractional conversion to the Ca2ϩ-bound state vs. the logarithm of the free-Ca2ϩ S65T͞H148D (22), some of the circularly permuted EGFPs Ϫ concentration in molarExRai-AktAR units. (D)Titrationsofnormalizedfluorescence would offer excitation ratioing and might avoid the Cl ion vs. pH with EDTA and zero Ca2ϩ,orwithsaturatingCa2ϩ.Themidpoints interference (20) and relatively easy photoisomerization (6) of Maltose Dopamine 2ϩ of the titration curves areVoltage 10.1 in EDTA and 8.9 in excess Ca . the current high-pKa EYFPs. MBP.PPYF (LL) dLight1 (LT) Tetsuya Yulong Li (YL) ASAP3 (ML) Kitaguchi (TK) Glutamate GRABDA(YL) calmodulin RS20 Among many others… cpGFP Pericam Glucose Atsushi Miyawaki iGluSnFR (LL) SF-iGluSnFR (LL) Green Glifons (TK) (RIKEN) …and from the the GCaMP1+ GCaMP3-7 GABA cAMP Junichi Nakai Loren Looger & Campbell lab: iGABASnFr (LL) Flamindo2 (TK) (Saitama) GENIE (Janelia) Jin Zhang (JZ) Michael Lin (ML) GINKO (K+) Nicotine cGMP iLACCO (lactate) Takeharu Nagai iNicSnFr (LL) Green cGull (TK) (Osaka) Citrate biosensors Nagai et al., Nat. Biotechnol. 2000, 18, 313; Nakai et al., Nat. Biotechnol. 2001, 19, 137; Extracellular ATP Intracellular ATP Tallini et al., Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4753; Wang et al., Structure 2008, 16, 1817; Akerboom et al., J. Biol. Chem. 2009, 284, 6455; iATPSnFr (LL) MaLionG (TK) Tian et al., Nat. Methods 2009, 6, 875; Akerboom et al., J. Neurosci. 2012, 32, 13819; Chen et al., Nature 2013, 499, 295; Dana et al., Nat. Methods 2019, 16, 649. 34 Why L-lactate? iLACCO: Astrocyte-to-Neuron Lactate Shuttle (ANLS) process A single FP-based L-lactate biosensor

OH O HO HO Efficient but slow OH OH Fast but inefficient production of ATP production of ATP (32/glucose) Glycolysis (2/glucose)

Figures O Aerobic O Anaerobic O - - Kreb’s CoA O O O (Fermentation) OH cycle Acetyl- Figures Figure 1. Design and in vitro characterizationCoenzyme of KIRIN1 A Pyruvate (a) Design andLactate construction ​ ​ ​ Figure 1. Design and in vitro characterization of KIRIN1 (a) Design and construction of the genetically encoded K+ indicator KIRIN1. mCerulean3 (residues 1-228) at the ​ ​ ​ ​ of the genetically encoded K+ indicator KIRIN1. mCerulean3 (residues 1-228) at the ​ “Lactate is transferred from astrocytes to neurons to match the neuronal energetic needs, and to provide There is growing recognition that lactate is a key player in a signals that modulate neuronal functions, including excitability, plasticity and memory consolidation.“ N-terminus is linked with Kbp (residues 2-149), followed by cp173Venus (full length) at N-terminus is linked with Kbp (residues 2-149), followed by cp173Venus (full length) at surprising variety of biological process (including neural activity). -Magistretti and Allaman the C-terminus. (b) Schematic demonstration of the molecular sensing mechanism of the C-terminus. (b) Schematic demonstration of the molecular sensing mechanism ofMagistretti and Allaman, Nature Reviews Neuroscience, , 19, 235. ​ ​ ​ ​ 2018 KIRIN1. Binding of K+ induces a structural change in the Kbp protein, increasing the KIRIN1. Binding of K+ induces a structural change in the Kbp protein, increasing the ​ ​ FRET efficiency between mCerulean3 and cp173Venus in KIRIN1. (c) KIRIN1 emission ​ ​ FRET efficiency between mCerulean3 and cp173Venus in KIRIN1. (c) KIRIN1 emission ​ ​ fluorescence spectrum (excitation 410 nm, emission 430 nm to 650 nm) in Tris HCl

+ fluorescence spectrum (excitation 410 nm, emission 430 nm to 650 nm) in Tris HCl buffer with (red) and without (blue) K .​ (d) FRET acceptor-to-donor fluorescence ratio ​ ​ ​

+ + buffer with (red) and without (blue) K .​ (d) FRET acceptor-to-donor fluorescence ratio (F530/F475) of purified KIRIN1 protein at various concentrations (1 µM to 150 mM) of K ​ ​ ​ ​ ​ ​ ​ ​ (red) and Na+ (blue) solutions. (e) FRET acceptor-to-donor fluorescence ratio of purified + ​ 35 ​ ​ 36 (F530/F475) of purified KIRIN1 protein at various concentrations (1 µM to 150 mM) of K ​ ​ ​ ​ Laconic is a currently available ​ KIRIN1 protein upon addition of various physiologically relevant ions including Mg2+ (10 Designs of fluorescent protein-based biosensors​ + (red) and Na (blue) solutions. (e) FRET acceptor-to-donorFRET-based biosensor for L-lactate fluorescence ratio of purified 2+ 2+ ​ ​ ​ mM), Ca ​ (10 µM), and Zn (10​ µM). ​ ​ Förster resonance energy transfer Fluorescent protein reconstitution KIRIN1 protein upon addition of various physiologically relevant ions including Mg2+ (10 ​

2+ 2+ mM), Ca ​ (10 µM), and Zn (10​ µM). ​ ​ LldR lactate- binding domain Split FPs

lactate A Y B A B Single fluorescent protein mTFP1 Venus FRET Dimerization-dependent FPs

>1000 works cited! Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX pubs.acs.org/CR

Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks Eric C. Greenwald,† Sohum Mehta,*,† and Jin Zhang*,† †University of California, San Diego, 9500 Gilman Drive, BRFII, La Jolla, CA 92093-0702, United States Miyawaki et al., Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2135; Ghosh et al., J. Am. Chem. Soc. 2000, 122, 5658; Kerppola, Chem. Soc. Rev.*S Supporting 2009 Information, 38, 2876; Nagai et al., Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3197; Nakai et al., Nat. Martin et al. PLoS ONE, 2013, 8, e57712. Biotechnol. 2001ABSTRACT:, 19,Cellular 137; signaling Alford networks et al., are theChem. foundation Biol. which 2012 determines, the19, fate 353; Ding et al., Nat Methods 2015, 12, 195. and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we 22 endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.

CONTENTS 3.4. How Fast Can We Go? AO 4. Obtaining Spatial Information from Biosensors AP 1. Introduction B 4.1. Membrane-Associated Signaling Compart- 2. Designing Genetically Encoded Biosensors B ments AR 2.1. Elements of Genetically Encoded Biosensors B 4.1.1. Partitioning by Membranes AR 2.2. Biosensors for Monitoring Protein Behavior M 4.1.2. Partitioning within Membranes AT 2.2.1. Protein Expression, Turnover, And Lo- 4.2. Compartmentation by the Activity of Path- calization M way Regulators AT 2.2.2. Protein−Protein Interactions M 4.2.1. Calcium Signaling AT 2.3. Biosensors for Monitoring Biochemical 4.2.2. cAMP Signaling AV Activity Dynamics N 4.2.3. Other Signaling Molecules AX 2.3.1. Inducing the Translocation of a Fluo- 4.3. Assembly of Macromolecular Signaling rescent Protein N Complexes AY 2.3.2. Directly Sensitizing the Chromophore Downloaded via UNIV OF ALBERTA on December 20, 2018 at 04:55:15 (UTC). 4.3.1. Scaffolding by A-Kinase Anchoring of a Fluorescent Protein P Proteins AY 2.3.3. Engineered Modulation of the Photo- 4.3.2. Molecular Scaffolds in Other Signaling See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. physical Behavior of a Fluorescent Pathways AZ Protein R 5. Computational Models and Fluorescent Biosen- 2.3.4. Coupled Reporter Systems AG sors AZ 2.4. New Biosensor Classes AG 5.1. Computational Modeling Background AZ 2.4.1. Sensors Based on Infrared Fluorescent 5.2. Examples of the Integrated Approach BB Proteins AG 5.2.1. Analysis of Temporal Dynamics BB 22 2.4.2. Biochemical Activity Integrators As in 5.2.2. Evaluation of Spatial Compartmentali- Vivo Snapshot Reporters AH zation BB 2.4.3. Fluctuation-Based Biosensors AI 5.2.3. Analyses of Cellular Heterogeneity BD 3. Obtaining Temporal Information from Biosen- 5.2.4. Information Theory BD sors AJ 6. Pushing the Field Forward BE 3.1. Kinetics of Individual Reactions AL 6.1. Improved Resolution BE 3.2. Higher-Order Signaling Dynamics AM 6.2. Multiplexed Biosensor Imaging BF 3.2.1. Adaptive Responses AM 3.2.2. Oscillations AN 3.2.3. Bistability and Ultrasensitivity AN 3.3. Importance of Single-Cell Readouts AO Received: May 25, 2018

© XXXX American Chemical Society A DOI: 10.1021/acs.chemrev.8b00333 Chem. Rev. XXXX, XXX, XXX−XXX 37 38 Our goal: an intensiometric lactate biosensor Step 1: Screen insertion sites

N-LBP cpGFP C-LBP cpGFP Screening of 70 different cpGFP insertion sites in loops cpGFP L-lactate N-LBP = residues 1 to X cpGFP C-LBP = residues X+1 to 361

Change in fluorescence +/- lactate (ΔF/F0 (%)) (%) 0 cpGFP L-lactate-binding protein (LBP) Δ F / LBP = Periplasmic Calcium L-Lactate- Binding Protein (TTHA0766) from Thermus thermophilus HB8

Yusuke Nasu (University of Tokyo) Insertion site (X) Footnotes Yusuke Nasu (University of Tokyo)

39 40 Step 2: Optimize linkers Step 3: Directed evolution ✔ ✔ +/- 0.8 The Nobel Prize in Chemistry cpGFP ✗ ✗ 2018 was one half awarded to DNA encoding prototype biosensor cpGFP Frances H. Arnold “for the Random mutations are The genes are inserted in bacteria, which use introducedRandom mutations in the gene are for the The genes are inserted enzyme that will be changed. 2 them as templates and directed evolution of enzymes.” 1 introduced in thegene gene for the in bacteria, which use produce randomly 1enzyme that will be changed. 2 them as templates and mutated enzymes. DNA produce randomly protein library

Fluorescence library mutated enzymes. DNA mutation 108 variants mutation

(%) enzymes 0 Optimize Wavelength enzymes

Δ F / this linker +/- 1.2 mutation ✗ ✔ mutation

cpGFP The changed enzymes are tested.The changed Those that enzymes are are 3 mosttested. efficient Those at that catalysing are 3themost desired efficient chemical at catalysing reactionthe desired are selected. chemical

Fluorescence reaction are selected. test plate screeningtest plate 128 variants repeat (%) 0 Wavelength multiple Optimize Optimized, higher-performance biosensor Δ F / times “losers” this linker +/- 1.8 - Larger fluorescence changes discarded - High folding efficiency enzymediscarded ✔ ✔ enzyme cpGFP - Brighter fluorescence - Optimized ligand affinity

New random mutations are introducedNew random in the mutations genes for are the improved

Fluorescence 4 selected enzymes. The cycle introduced in the genes for the 4beginsselected again. enzymes. The cycle “winners” begins again. Yusuke Nasu (University of Tokyo) Wavelength ©Johan Jarnestad/The Royal Swedish Academy of Sciences ©Johan Jarnestad/The Royal Swedish Academy of Sciences 41 42 Step 3: Directed evolution iLACCO1 crystal structure ✔ ✔ cpGFP X-ray crystal structure of iLACCO1 DNA encoding prototype biosensor Random mutations are The genes are inserted in bacteria, which use introducedRandom mutations in the gene are for the The genes are inserted enzyme that will be changed. 2 them as templates and 1 introduced in the gene for the in bacteria, which use gene produce randomly 1enzyme that will be changed. 2 them as templates and mutated enzymes. DNA produce randomly protein library Directed evolution library mutated enzymes. iLACCO1 DNA mutation (7 rounds in total) mutation enzymes enzymes

mutation Kd = 96 µM

0 mutation

The changed enzymes are tested.The changed Those that enzymes are are Δ F / 3 mosttested. efficient Those at that catalysing are 3themost desired efficient chemical at catalysing reactionthe desired are selected. chemical reaction are selected. ΔF/F0: ~6 test plate screeningtest plate C-Linker optimization N-Linker optimization 7x + L-lactate “losers” - L-lactate discarded Fluorescence intensity enzymediscarded 2+ iLACCO variants tested enzyme Unfortunately, L-lactate binding is [Ca ]-dependent, so Wavelength (nm) Wavelength iLACCO1 does not work intracellularly. Fortunately, our aim 2+ New random mutations are improved was to image L-lactate extracellularly, where [Ca ] is high. introducedNew random in the mutations genes for are the 4 selected enzymes. The cycle introduced in the genes for the 4beginsselected again. enzymes. The cycle “winners” begins again. Yusuke Nasu (University of Tokyo) ©Johan Jarnestad/The Royal Swedish Academy of Sciences Yusuke Nasu with Shuce Zhang and Dr. Yurong Wen in lab of Dr. Joanne Lemieux (Alberta) ©Johan Jarnestad/The Royal Swedish Academy of Sciences

43 iLACCO1.1 (optimized for extracellular L-lactate)

L-lactate HeLa cells + 10 mM L-lactate

K = 1.1 mM d Redder is Better! Extracellular side

Intracellular side Advantages of red-shifted biosensors

✔ Tuned affinity to respond to Imaging deeper in non-transparent animals extracellular concentration range Less phototoxicity for long-term observations ✔ Glycophosphatidylinositol (GPI) Multicolour, multi-parameter imaging anchor for extracellular display ✔ Optimized linker between GPI anchor Use with optogenetic stimulation (e.g., ChR2) and biosensor ✔ Non-lactate binding control version

Yusuke Nasu with Qi Dong (University of Tokyo) 45 46 Redder is better (for imaging deeper into tissue) Redder is better (for imaging deeper into tissue)

1P excitation 2P excitation 1P excitation 2P excitation The near-infrared window is The near-infrared window is the range of wavelengths in the range of wavelengths in 10 Near-infrared window (~650 10 Near-infrared window (~650 which tissue is most which tissue is most nm ~ 1350 nm) nm ~ 1350 nm) transparent to light. transparent to light. 1 1 Scattering of light by tissue Scattering of light by tissue decreases with increasing decreases with increasing 0.1 Deoxygenated blood 0.1 Deoxygenated blood wavelength. wavelength.

Relative absorbance Relative 0.01 Ideally, neural activity absorbance Relative 0.01 Ideally, neural activity Oxygenated blood Oxygenated blood biosensors (Ca2+, voltage, etc.) biosensors (Ca2+, voltage, etc.) would absorb and fluoresce in would absorb and fluoresce in 400 600 800 1000 1200 1400 this region 400 600 800 1000 1200 1400 this region 1600 1600 Wavelength (nm) Wavelength (nm) Aequorea- derived FPs Adapted from omlc.org Adapted from omlc.org

47 48 Redder is better (for imaging deeper into tissue) Four classes of fluorescent protein Biliverdin-binding proteins Homologs of Aequorea GFP 1P excitation 2P excitation with autogenic The near-infrared window is chromophore formation the range of wavelengths in 10 Near-infrared window (~650 which tissue is most O nm ~ 1350 nm) N N transparent to light. O O HO N Coral & anemone FPs 1 H Jellyfish FPs (e.g. GFP) (e.g. mCherry) Scattering of light by tissue decreases with increasing 0.1 Deoxygenated blood Flavin-binding proteins Bilirubin-binding proteins Phytochrome-derived wavelength. (e.g. IFP1.4, iRFP)

Relative absorbance Relative 0.01 Ideally, neural activity Oxygenated blood biosensors (Ca2+, voltage, etc.) O O O O would absorb and fluoresce in NH HN O NH HN N this region NH NH N 400 600 800 1000 1200 1400 NH HN 1600 N N O HOOC COOH Wavelength (nm) HOOC COOH R Aequorea- Coral-derived flavin-binding protein-derived fatty-acid-binding protein-derived Allophycocyanin-derived derived yellow/orange/ (e.g. iLOV) (e.g. UnaG) (e.g. smURFP) FPs red FPs Shu et al., Science 2009, 324, 804 (IFP1.4); Filonov et al., Nat. Biotechnol. 2011, 29, 757 (iRFP); Yu et al., Nat. Methods 2015, 12, 763 (mIFP); Adapted from omlc.org Rodriguez et al. Nat. Methods 2016, 13, 763 (smURFP); Kumagai et al., Cell 2013, 153, 1602 (UnaG); Buckley et al., Curr. Opin. Chem. Biol. 2015, 27, 39 (iLOV). 49 50 Engineering of red Ca2+ biosensors K-GECO1: an improved red Ca2+ biosensor Improved Rational engineering & performance directed evolution R-CaMP2 … which led us to develop a new biosensor …and recently we’ve made a jRGECO1a based on an improved fluorescent protein highly red shifted variant XCaMP-R DOES NOT New colors Bubble-tip EXPRESS 2011 - 2019 O-GECO anemone WRONG CAR-GECO AFFINITY REX-GECO TOO Ca2+ DIM biosensor Affinity tuning for ER and mito ? LAR-GECO + R-GECO1 R-CEPIA1er DOES Ca2+ biosensor K-GECO1 NOT RESPONSE and others… RESPOND TOO WEIRD COMPLEX SMALL PHOTO- PHOTO- PHYSICS PHYSICS mNeptune However, we have found some problems MIS-LOCALIZES IN mCardinal mKelly with this particular red FP domain… CELLS mKate2* variant (FusionRed) Zhao et al., Science 2011, 333, 1888 (R-GECO1); Wu et al., ACS Chem. Neurosci. 2013, 4, 963 (O-GECO, R-GECO1.2); Wu et al., Nat Commun 2014, 5, 5262 (REX-GECO); Yi Shen Wu et al., Biochem. J. 2014, 464, 13 (LAR-GECO); Ohkura et al., PLoS ONE 2012, 7, e39933 (R-CaMP1.07); Inoue et al., Nat Methods 2015, 12, 64 (R-CaMP2); Yongxin Zhao and Jiahui Wu Suzuki et al., Nat Commun 2014, 5, 4153 (R-CEPIAer); Dana et al. eLife 2016, 5, e12727 (jRCaMP1ab, jRGECO1a); Inoue et al., Cell 2019, 177, 1346 (XCaMP-R). Shemiakina et al., Nat. Commun. 2012, 3, 1204 (mKate2 variant FusionRed); Shen et al., BMC Biology 2018, 16, 9 (K-GECO1).

51 52 AnalyticalConverting other biosensors from green to red Chemistry Article Redder is better (for imaging deeper into tissue)

“Transplanting” red FP FlicR1 R-iGluSnFR1 domain of R-GECO1 into 1P excitation 2P excitation existing green biosensors The near-infrared window is the range of wavelengths in 10 Near-infrared window (~650 which tissue is most nm ~ 1350 nm) transparent to light. 1 Scattering of light by tissue R-GECO1 decreases with increasing voltage glutamate 0.1 Deoxygenated blood Ca2+ biosensor wavelength.

Relative absorbance Relative 0.01 Ideally, neural activity Plus a growing selection of red FP Oxygenated blood 2+ biosensors from other labs… biosensors (Ca , voltage, etc.) would absorb and fluoresce in 400 600 800 1000 1200 1400 this region Wavelength (nm) 1600 MaLionR ATP Aequorea- Coral-derived ZnRed Zn2+ biosensor biosensor Pink Flamindo cAMP biosensor Figure 1. Engineering of single-FP based GEZIs. (A) Domain arrangements of various constructs. Important linker sequences are shown in derived yellow/orange/ ff individual boxes, and mutations within the FP sca old are also indicated. TheJin fullet al., amino Neuron acid 2012 sequences, 75, 779 of (Arclight); these constructs Abdelfattah are et al., available J. Neurosci. in Figures 2016, S1 36, 2458 (FlicR1); Marvin et al., Nat. Methods 2013, 10, 162 (iGluSnFr); FPs red FPs and S2. (B−D) Fluorescence excitation (open circle) and emission (filled circle) spectra of ZnGreen1 (B), ZnGreen2 (C), and ZnRed (D), in the Wu et al., ACS Chem. Biol., 2018, Article ASAP (R-iGluSnFR1); Chen and Ai, Anal. Chem. 2016, 88, 9029 (ZnRed); Harada2+ et al., Sci. Rep. 2017, 7, 7351 (Pink Flamindo); Arai et al., Angew. Chem. IE 2018, 57, 10873 (MaLionR). Adapted from omlc.org presence (gray line, treated with 100 μM ZnCl2) and absence (black line, treated with 100 μM EDTA) of Zn .

nm HeNe laser, and the red fluorescence emission was based GEZIs.19,33 In a similar strategy to the insertion of a collected between 560 and 700 nm with a hybrid detector Ca2+-binding calmodulin domain into a FP, as in “Camgaroo” (HyD). Time-lapse series was acquired at 1 min interval and indicators,29 we reasoned that inserting Zap1 zinc fingers into fluorescence emissions from both channels were scanned FPs would be a promising strategy to create GEZIs. For the sequentially with the “between lines” mode. Following a few fluorescent element, we selected monomeric teal fluorescent initial image acquisitions, Zn2+/ pyrithione/KHB (50 μM Zn2+ protein (mTFP1) because of its high fluorescence brightness, and 5 μM pyrithione) solution was added, and the time series excellent photostability, and efficient chromophore maturation continued for another 10 min. at 37 °C.34 Directly inserting Zap1 into the β-barrel nearest the To stimulate zinc release in INS-1E cells, transfected cells phenolate chromophore of mTFP1 would disrupt the fi expressing pDisplay-ZnGreen1 were rst incubated with 10 μM chromophore folding and subsequently complicate attempts EDTA/KHB solution for 15 min to remove prebound metals. to rescue fluorescence loss caused by this insertion. Thus, to Cells were then washed three times with and then maintained make the β-barrel more tolerable to modifications, we first ff in KHB bu er to remove EDTA before imaging. Cells were created an efficiently folded, highly fluorescent circularly imaged on the confocal SP5 microscope. ZnGreen1 was excited fl permutated version of mTFP1. We fused the original N- and with a 488 nm argon-ion laser and green uorescent emission C-terminal ends with a GGTGGS hexapeptide linker and split was collected between 495 and 600 nm with a hybrid detector mTFP1 near the phenolate chromophore between residues 144 (HyD). Following the initial four image acquisitions, INS-1E and 145 (Figure 1A). Because the resultant recombinant cpFP cells were stimulated with 35 mM glucose/KHB solution and fl fl (circularly permuted FP) was essentially non uorescent, we time-lapse uorescence imaging continued for another 20 min. then used degenerate codons to extend both the new N- and C- All images and videos were processed and rendered with termini in order to improve the fluorescence. Screening the ImageJ. resultant library yielded a moderately improved fluorescent clone, cpTFP0.5, which harbored extra N- and C- terminal ■ RESULTS AND DISCUSSION extensions (Figure S1). We next performed five successive Development of Green Fluorescent Zn2+ Indicators. rounds of directed evolution by using error-prone PCR- The first and second zinc fingers of Saccharomyces cerevisiae generated libraries and screened the resultant bacterial colonies transcription factor, Zap1, is an ideal proteinaceous, Zn2+- after overnight incubation at 37 °C for improved fluorescence binding domain for constructing GEZIs due to their small size, brightness. This effort led to cpTFP1, which harbors five high Zn2+-binding affinity, and Zn2+-induced conformational additional mutations (N42H, N81D, S146P, R149 K, and E168 change, and it has been successfully exploited to derive FRET- K; residues numbered according to the numbering of mTFP1

9031 DOI: 10.1021/acs.analchem.6b01653 Anal. Chem. 2016, 88, 9029−9036 53 54 Redder is better (for imaging deeper into tissue) Four classes of fluorescent protein Biliverdin-binding proteins Homologs of Aequorea GFP 1P excitation 2P excitation with autogenic The near-infrared window is chromophore formation the range of wavelengths in 10 Near-infrared window (~650 which tissue is most O

nm ~ 1350 nm) N N transparent to light. O O HO N Coral & anemone FPs 1 H Jellyfish FPs (e.g. GFP) (e.g. mCherry) Scattering of light by tissue decreases with increasing 0.1 Deoxygenated blood Flavin-binding proteins Bilirubin-binding proteins Phytochrome-derived wavelength. (e.g. IFP1.4, iRFP)

Relative absorbance Relative 0.01 Ideally, neural activity Oxygenated blood biosensors (Ca2+, voltage, etc.) O O O O would absorb and fluoresce in NH HN O NH HN N 400 600 800 1000 1200 1400 this region NH NH N 1600 NH HN N N O HOOC COOH Wavelength (nm) HOOC COOH R Aequorea- Coral-derived Biliverdin- flavin-binding protein-derived fatty-acid-binding protein-derived Allophycocyanin-derived derived yellow/orange/ binding near- (e.g. iLOV) (e.g. UnaG) (e.g. smURFP) FPs red FPs infrared FPs Shu et al., Science 2009, 324, 804 (IFP1.4); Filonov et al., Nat. Biotechnol. 2011, 29, 757 (iRFP); Yu et al., Nat. Methods 2015, 12, 763 (mIFP); Adapted from omlc.org Rodriguez et al. Nat. Methods 2016, 13, 763 (smURFP); Kumagai et al., Cell 2013, 153, 1602 (UnaG); Buckley et al., Curr. Opin. Chem. Biol. 2015, 27, 39 (iLOV).

55 56 Engineering a NIR Ca2+ biosensor mIFP-derived NIR-GECO1

Rational engineering & directed evolution

DOES Absorbance and emission 2+ NOT peaks at 678 nm and 704 Ca EXPRESS nm, respectively WRONG AFFINITY TOO mIFP DIM

+ NIR-GECO1 - Ca2+ 1.2 NIR-GECO1 + Ca2+ e c

n 1.0 e c s

DOES ~10-fold decrease in e r 0.8 Kd = ~200 nM o u

NOT l f RESPONSE fluorescence intensity 0.6 d

RESPOND WEIRD e

TOO z i

NIR-GECO1 2+ l 0.4 Calmodulin SMALL PHOTO- upon binding Ca a m PHYSICS r 0.2 o

RS20 N + 0.0 550 600 650 700 750 800 Wavelength (nm) Yong Qian Yong Qian Yu et al., Nat. Methods 2015, 12, 763 (mIFP); Qian et al., Nat. Methods, 2019, 16, 171 (NIR-GECO1). Yu et al., Nat. Methods 2015, 12, 763 (mIFP); Qian et al., Nat. Methods, 2019, 16, 171 (NIR-GECO1).Footnotes 57 58 Multi-colour & multi-parameter imaging Redder is Better!

NIR-GECO1 Ca2+ (Ca2+)

Ca2+ Pink Flamindo (cAMP)

Advantages of NIR-GECO1 AKAR4 Imaging deeper into tissue (Protein kinase A) ✔Multicolour imaging ✔Use with optogenetic actuators Sohum Mehta and Jin Zhang Yu et al., Nat. Methods 2015, 12, 763 (mIFP); Qian et al., Nat. Methods, 2019, 16, 171 (NIR-GECO1).Footnotes

59 60 Progress towards NIR-GECO2 Summary of single FP-based biosensors

1.0 “Redder is better”

NIR-GECO1.XNIR-GECO2 - Growing selection of RFP-derived Higher NIR-GECO1NIR-GECO1 biosensor. affinity 0.5 - mIFP-derived NIR-GECO1 is 1st ~100 nM generation NIR Ca2+ biosensor - Future: 2nd generation NIR- 0.0 Normalized fluorescence -2 -1 0 1 2 GECO1+ with improved sensitivity for in vivo imaging.

iLACCO1.1 lactate biosensor Brighter - iLACCO1.1 is a 1st generation single FP-based biosensor for ~2x extracellular lactate Spontaneous neuronal spiking in dissociated neurons - Future: Imaging of lactate released by astrocytes and new

Fluorescence (a.u.) expressing NIR-GECO1.X biosensors for intracellular lactate imaging.

NIR-GECO1 Works great in worms! NIR-GECO1.X Genes from www.addgene.org/Robert_Campbell/ -Ed Boyden, Kiryl Piatkevich Yong Qian and Kiryl Piatkevich Contact: [email protected] or [email protected] 61 Summary Dr. Eason Shen (K-GECO; FR-GECO; GINKO) Dr. Yufeng Zhao (Citrate biosensor) Sally Wu (GINKO) Fluorescent proteins (FPs) Dr. Yong Qian (NIR-GECO) Other fluorophore technologies Rochelin Dalangin (FR-GECO) Single FP-based biosensors

Take-home messages: 1. Despite “optimization” no one FP or biosensor is ideal by all criteria, and it is typically impossible to predict which one will work best in a new application. 2. I recommend trying 2-3 different FPs or biosensors (from different species), and determining which one is best under your experimental conditions. 3. All other factors being the same, redder is better. Assistant Professor 4. There is a growing selection of intensiometric single FP-based biosensors, and methods Dr. Yusuke Nasu (iLACCO) for developing new ones are becoming increasingly well-established.

63 !64 Acknowledgments Dr. Yong Qian (NIR-GECO1+); Sheng-Yi (Sally) Wu; Shuce Zhang; Rochelin Dalangin; Xiaocen Lu; Dr. Yi Shen; Dr. Currently looking to Yufeng Zhao; Dr. Fang Zheng; Dr. Landon Zarowny; Yan Li hire a Project Assistant Assistant Professor Dr. Yusuke Nasu (lactate); Rina Professor in Tokyo. Hashizume; Peter Wojnicki; Hayato Kadoya; and Applications welcome! undergraduate summer interns. Alumni Landon Zarowny, Ph.D.; Wei Zhang, Ph.D.; Matthew Wiens, Ph.D.; Ahmed Abdelfattah, Ph.D.; Tiffany Yan Lai, M.Sc.; Jhon Ralph Enterina, M.Sc.; Yi Shen, Ph.D.; Jiahui Wu, Ph.D.; Nazanin Assempour, M.Sc.; Yan Li, M.Sc.; Yongxin Zhao, Ph.D.; Yidan Ding, Ph.D.; Ahmed Belal, Ph.D.; Haley Carlson, Ph.D.; Hiofan Hoi, Ph.D.; Ritesh Saini, M.Sc.; Spencer Alford, Ph.D.; Andreas Ibraheem, M.Sc.; Zihao Cheng, Ph.D.; Hui-wang Ai, Ph.D.; Carine Lafaille, M.Sc.; Yankun Li, M.Sc.; Dr. Tam Tran; Dr. Yingche Chen; Dr. Cory Beshara; Dr. Hongkin Yap; Dr. Monika Johar; Aillette Sierra Mulet, and many undergraduates.

Collaborators for work shown Kiryl Piatkevich (NIR-GECO1) & Ed Boyden (MIT) Sohum Mehta (NIR-GECO1) and Jin Zhang (UCSD) Yurong Wen (X-ray structure) & Joanne Lemieux (Alberta)

Support for this work NIH (BRAIN Initiative with JB Pierce and MSU) Alberta Innovates (Scholarships) Brain Canada (latform and NIH matching) University of Alberta NSERC (Discovery grant and scholarships) University of Tokyo and GSC program CIHR (Foundation) JSPS Kakenhi (S) 2019-2023