Objectives for today: Why targeted and expressible probes Aequorin & GFP mixed with theory

1. PBio/NeuBehav 550: Biophysics of Ca2+ signalingWeek 2 (04/08/13)Genetically expressible probes and FRET Objectives for today: Why targeted and expressible probes Aequorin & GFP mixed with theory FRET Theory and photochemistry The first cameleons Discuss the 2nd generation cameleon paper

2. Standard tools for calcium studies Tools for calcium studies [Caged calcium] [NP-EGTA] [–—NP] The original Ca/Mg chelator & buffer EDTA (1946) EGTA (1955) BAPTA (1980) Fura, Indo Ca Green Ca-selective chelator & buffer slow, pH sensitive Roger Tsien’s fast buffers & fluorescent indicators KCa ~ 80-300 nM

3. Ca2+ fluxes in an excitable cell Typical Ca2+ fluxes in a non-excitable cell Inputs: hormones, cytokines, growth factors, antigens PIP2 Agonist Na+-Ca2+ exchanger R DAG Gq PLC IP3 LDCSG Ca2+ Ca2+ Ca2+ IP3R channel SERCA pump Na+ ATP nucleus ER Plasma membrane Ca2+ ATP Ca2+ Mito PM Ca2+ ATPase SOC/CRAC channel Ca2+ Na+ Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

4. Proteins as indicators Advantages of proteins as indicators Highly evolved binding sites Can be further engineered by mutation Sophisticated optical properties Expressed by transfection, infection, transgenic; no loading; do not leak Targetable to: specific cell types at specific times in organisms subcellular locations and organelles in cells

5. Genetic targeting of fluorescent constructs Targeting Targeted to: cytoplasm N C fluorescent protein ER KDEL CRsig fluorescent protein secretory granules fluorescent protein tpA nucleus nls fluorescent protein mitochondria COX8 fluorescent protein Abbreviations: CRsig = calreticulin signal sequence KDEL = ER retention signal tpA = tissue plaminogen activator (a secreted protein) nls = nuclear localization signal COX8 = cytochrome oxidase N-terminus

6. Localization Targeting of fluorescent proteins scales = "10 mm" YC2 nuGFP and mtBFP YC3er (Ruzzuto et al. & Tsien, Nature, 1996) (Miyawaki et al. & Tsien, Nature, 1997)

7. Aequoria Fluorescent proteins make Aequorea glow at 508 nm The Nobel Prize in Chemistry 2008. Osamu Shimomura, Martin Chalfie, Roger Y. Tsien Green fluorescent ring ---Shimomura O, Johnson FH, Saiga Y, 1962, Extraction, purification and properties of Aequorin, a biolumi-nescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol., 59: 223-239. [470 nm] ---R.Y. Tsien, 1998, The Green FluorescentProtein, Annual Review of Biochemistry 67, pp 509-544. [508 nm] Aequorea victoria from Puget Sound in brightfield and false color

8. Aequorin 2 Aequorin: a bioluminescent Ca2+ binding protein complex containing coelenterazine coelenterazine M.W. = 22,514 with four E/F hands Aequorin (Aeq) falls in the general heading of "luciferases" that bind a "luciferin" and luminesce in response to a ligand. (The most famous of these is firefly luciferase that can be used to measure ATP concentrations.) Reaction: Aeq + coelenterazine ----> Aeq.c [non-covalent complex] Aeq.c + ~3 Ca2+ ----> Ca3.Aeq.c* + CO2 Ca3.Aeq.c* -----> Ca3.Aeq.c** + [blue photon--470 nm] Aequorin is therefore a one-shot calcium detector with a non-linear Ca2+ dependence of luminescence. It is "consumed" by a detection event.

9. Ca2+ fluxes in an excitable cell Stimulating a Ca2+ signal in cytosol & mitochondria Inputs: hormones, cytokines, growth factors, antigens PIP2 Agonist e.g. histamine Na+-Ca2+ exchanger R DAG Gq PLC IP3 LDCSG Ca2+ Ca2+ Ca2+ IP3R channel Na+ SERCA pump ATP ER Plasma membrane Ca2+ ATP Ca2+ Mito PM Ca2+ ATPase SOC/CRAC channel Ca2+ Na+ Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

10. Biological example aequorin Targeted aequorin reports [Ca] in mitochondrial matrix protonophore FCCP depolarizes inner membrane of mitochondrion Aeq targeted inside mitochondrial matrix Δψ histamine stimulus 10 cytoplasmic Ca is sucked into mitochondria by Δψ reported [Ca] (mM) 5 Control test: with 5 mM FCCP, Ca does not enter HeLa cells transfected with an aequorin construct targeted all the way into the matrix of mitochondria. Cells were then soaked in micromolar coelenterazine at zero calcium for several hours. (Rizzuto...Pozzan, Science, 1998)

11. Why are most proteins not visibly fluorescent? coelenterazine emits 470 nm Tyrosine/ phenol: Excit. 275 nm, emits 310 nm) napthalene anthracene tetracene "Particle-in-a-box" (think organ pipes) absorption spectra UV visible small box, short wave large box, long wave

12. GFP GFP: generates a fluorescent chromophore from its amino acids autocatalytically Y66 G67 Maturation can be slow Engineer codons folding color photoconversion M.W. = 26,938 N dehydration C GFP, a beta barrel

14. Colored GFPs Engineering color in GFPs Excitation spectra Emission spectra 4 5 5 4 Absorbance Fluorescence intensity 300 400 500 600 400 500 600 700 wavelength (nm) wavelength (nm) Roger Tsien's lab made a range of GFP-derived proteins of different colors by mutation of the expression vector.

15. Absorption and fluorescence spectra reflect internal energy levels Absorption bands S1 S1 Energy S0 S0 ground state Jablonski diagram Absorption wavelength Absorber has several electronic states (S0, S1, S2, etc.). It also has vibrational states whose close spacing means that photons of a range of close energies can be absorbed. If the absorption spectrum has a second peak (at shorter wavelength), it is for excitation to S2 or because the dye has several molecular forms/conformations.

16. Förster/Fluorescence resonance energy transfer(FRET): A proximity detector (molecular ruler) that changes color FRET illustrate 440 nm 480 nm YFP hn CFP emission hn Separated: no FRET excitation no 440 nm excitation no hn 440 nm hn hn YFP FRET! CFP 535 nm Close together: FRET excitation emission Green fluorescent protein (GFP) has been engineered to make forms with various fluorescent colors (GFP, CFP, YFP, …). They have overlapping spectra and can transfer excitation directly by FRET when the proteins are close together. The energy transfer occurs without a photon.

17. Forster Eq FRET depends steeply on distance. R depends on overlap. Donor Acceptor 440 nm YFP FRET! CFP 535 nm excitation emission r fDeA Ro6 Ro6 + r 6 Transfer efficiency E: E = ------------- Förster formula for Förster radius Ro Ro = Const. {fdonk2J n –4} 1/6 Where fdon quantum efficiency of donor k orientation factor (0 – 4) n local refractive index J "overlap integral" of donor fluorescence (fD) and acceptor absorption eA J = 500 600 l = wavelength

18. More steps in the Jablonski diagram internal conversion (1 ps) (polar) solvent relaxation (100 ps) competition for re-radiation, quench, FRET, or other non- radiative (3 ns) absorption (1 fs) knr hnFRET fluorescence quench FRET Donor Acceptor

19. Lifetime & FRET FRET speeds donor F and slows acceptor F Ca2+-bound CaMeleon competition for re-radiation, quench, FRET (polar) solvent relaxation (100 ps) 530 nm from EYFP by FRET emission intensity internal conversion (1 ps) absorption (1 fs) knr 480 nm from ECFP hnFRET Donor Acceptor Fluorescence lifetime imaging is a way to image FRET quench fluorescence CFP FRET YFP 0 2 4 6 time (ns) Fluorescence decays recorded with YC3.1 cameleon dissolved in buffer. Excitation at 420 nm excites the ECFP part. (Habuchi et al. Biophys J, 2002)

20. FRET as a ‘Spectroscopic Ruler’ The efficiency of energy transfer is proportional to the inverse of the sixth power of the distance separating the donor and acceptor fluorophore ECFP/EYFP Förster distance 30 Å Förster distance 50 Å e.g., ECFP/EYFP Förster distance 70 Å E % decreases with the distance between donor and acceptor Two fluorophores separated by Förster distance (r = Ro) have E transfer of 50%

21. A family of Ca2+-sensitive switches and buffers Calmodulin helix-loop-helix makes E-F hand { x x x x KCa ~ 14 mM for free calmodulin Calmodulin MW ~ 17 kDa Calmodulin (CaM) : An abundant 149 amino acid, highly conserved cyto-plasmic protein with 4 binding sites for Ca2+ each formed by "EF-hands." Many other homologous Ca2+ binding proteins of this large EF-hand family act as Ca switches and Ca buffers. The Ca2+ ions bind cooperatively and become encircled by oxygen dipoles and negative charge. CaM com-plexes with many proteins, imparting Ca2+-dependence to their activities.

22. Calmodulin folds around a target helix Calmodulin folds MLCK peptide 4 Ca CaM Binding of Ca2+ to CaM causes CaM to change conformation. Binding of CaM to targets can increase the Ca2+ binding affinity of CaM greatly. The target peptide in this crystal structure is the regulatory domain of smooth-muscle myosin light-chain kinase (MLCK). The interaction of CaM and MLCK allows smooth muscle contraction to be activated in a Ca2+-dependent manner. (Meador WE, Means AR & Quiocho, 1992.)

23. Design of CaMeleons: Expressible proteins for Ca detection Design of CaMeleons: 440 nm 480 nm YFP Low calcium: No FRET C N CaM MLCK CFP C YFP 440 nm FRET CFP High calcium: FRET 535 nm N Two GFPs in one peptide interact by fluorescence resonance energy transfer (FRET). Targeting sequences can be added to direct constructs to specific compartments. (Miyawaki, Roger Tsien et al., 1997)

24. Ca-sensitive cameleon emission spectra Note two peaks no Ca Ca YC3.1 cameleon emission intensity more FRET Emission wavelength (nm) (Miyawaki, Roger Tsien et al., 1997)

25. Cameleon emission combines two spectra EYFP ECFP Ca no Ca YC3.1 cameleon emission intensity emission ECFP EYFP There is FRET even with no Ca2+! Amount of FRET gives distance changes. It is not a large change.

26. Ca-sensitive FRET reporter. How do calciums bind? (Miyawaki et al., 1997) green cameleon 1 fluorescence ratios 1.0 E104 C N E31 lower affinity higher affinity GC1 510/445 nm emission ratio GC1/E31Q GC1/E104Q free calcium (M) Calcium binding and the conformation change can be tailored by making mutations in the EF hand regions of the calmodulin. Glutamate E31 is in the first EF hand (at p12') and E104 is in the third EF hand (also at p12').

27. ER-directed Cameleon (Dickson,....,Hille, 2012) PC12 cells are transfected with D1-ER, a Roger Tsien cameleon directed to the ER. SERCA pump blocker BHQ shows efflux, ATP shows efflux with a transient refilling by outside Ca due to SOCE. ATP makes IP3 production,

28. Miyawaki et al. 1999 paperDynamic and quantitative Ca2+ measurements using improved cameleonsEach figure will be described by a student--as if you are teaching it to us for the first time. Further questions will come from the audience. --5 min per fig--one panel at a time --give it a title --explain axes and subject --ask leading questions to get students to discuss--what is being tested and what is concluded? Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin

29. Fig 1 G67 Y66 0.1 0.0 2.1 2 2.1 Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin 2

30. Fig 2AB YC2.1 2.1 3.1 Emission wavelength (nm) 3.1 2.1 Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin

31. Fig 2CD 3.1 Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin 2.1 2.1 3.1

32. Fig 3 YC2.1 YC2 Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin

33. Fig 4 YC2.1 500 uM 150 uM YC3.1 40 uM Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin

34. Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang Fig 5. Jerome Cattin Fig 5 CaM split 2.1 2.1 3.1 split 2.1 YC3.1 +- CaM Emission wavelength (nm)