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PBio/NeuBehav 550: Biophysics of Ca2+ signaling Week 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 Standard tools for calcium studies The original Ca/Mg chelator & buffer EDTA (1946) EGTA (1955) [NP-EGTA] BAPTA (1980) Fura, Indo Ca Green [–—NP] Ca-selective chelator & buffer slow, pH sensitive [Caged calcium] Roger Tsien’s fast buffers & fluorescent indicators KCa ~ 80-300 nM Typical Ca2+ fluxes in a non-excitable cell Inputs: hormones, cytokines, growth factors, antigens PIP2 Agonist R IP3R channel Plasma membrane DAG Gq PLC LDCSG IP3 Ca2+ Ca2+ ATP Na+-Ca2+ exchanger SERCA pump Ca2+ nucleus ER Ca2+ ATP Ca2+ Mito SOC/CRAC channel Na+ Ca2+ PM Ca2+ ATPase Na+ Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression 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 Genetic targeting of fluorescent constructs Targeted to: cytoplasm N fluorescent protein C ER CRsig fluorescent protein KDEL tpA fluorescent protein secretory granules nucleus mitochondria fluorescent protein COX8 nls 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 Targeting of fluorescent proteins YC2 scales = "10 mm" nuGFP and mtBFP YC3er (Miyawaki et al. & Tsien, Nature, 1997) (Ruzzuto et al. & Tsien, Nature, 1996) 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 bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol., 59: 223-239. [470 nm] Aequorea victoria from Puget Sound in brightfield and false color ---R.Y. Tsien, 1998, The Green Fluorescent Protein, Annual Review of Biochemistry 67, pp 509-544. [508 nm] 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* ----> Ca3.Aeq.c* + CO2 -----> 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. Stimulating a Ca2+ signal in cytosol & mitochondria Inputs: hormones, cytokines, growth factors, antigens Agonist e.g. histamine PIP2 R DAG Gq PLC IP3R channel LDCSG IP3 Ca2+ Ca2+ ATP Na+-Ca2+ exchanger Ca2+ SERCA pump Plasma membrane ER Ca2+ ATP Ca2+ SOC/CRAC channel Na+ Mito Ca2+ PM Ca2+ ATPase Na+ Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression Targeted aequorin reports [Ca] in mitochondrial matrix Aeq targeted inside mitochondrial matrix Δψ histamine stimulus protonophore FCCP depolarizes inner membrane of mitochondrion 10 5 cytoplasmic Ca is sucked into mitochondria Control test: by Δψ 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) Why are most proteins not visibly fluorescent? coelenterazine emits 470 nm Tyrosine/ phenol: Excit. 275 nm, emits 310 nm) napthalene anthracene "Particle-in-a-box" (think organ pipes) absorption spectra UV small box, short wave large box, long wave tetracene visible GFP: generates a fluorescent chromophore from its amino acids autocatalytically Y66 G67 Maturation can be slow Engineer codons folding color photoconversion dehydration M.W. = 26,938 N C GFP, a beta barrel Engineering color in GFPs Excitation spectra 5 4 5 400 500 Absorbance Fluorescence intensity 4 Emission spectra 300 400 500 wavelength (nm) 600 600 700 wavelength (nm) Roger Tsien's lab made a range of GFP-derived proteins of different colors by mutation of the expression vector. Absorption and fluorescence spectra reflect internal energy levels S1 S1 S0 S0 ground state Absorption wavelength Jablonski diagram 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. Förster/Fluorescence resonance energy transfer (FRET): A proximity detector (molecular ruler) that changes color 440 nm 480 nm hn emission hn Separated: excitation no FRET 440 nm Close together: FRET YFP CFP hn excitation no 440 nm excitation no hn CFP FRET! YFP hn 535 nm 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. FRET depends steeply on distance. R depends on overlap. Donor Acceptor 440 nm CFP FRET! YFP excitation r Transfer efficiency E: 535 nm emission R o6 E = ------------R o6 + r 6 fD eA Förster formula for Förster radius Ro Ro = Const. {fdon k2 J n –4} 1/6 Where fdon quantum efficiency of donor k orientation factor (0 – 4) n local refractive index 500 600 J "overlap integral" of donor fluorescence (fD) and acceptor absorption eA J= l = wavelength More steps in the Jablonski diagram internal conversion (1 ps) (polar) solvent relaxation (100 ps) competition for re-radiation, quench, FRET, or other nonradiative (3 ns) absorption (1 fs) knr fluorescence Donor hnFRET quench FRET Acceptor FRET speeds donor F and slows acceptor F competition for re-radiation, quench, FRET (polar) solvent relaxation (100 ps) internal conversion (1 ps) absorption (1 fs) emission intensity Donor knr fluorescence CFP hnFRET quench Acceptor FRET YFP Ca2+-bound CaMeleon 530 nm from EYFP by FRET 480 nm from ECFP 0 2 time (ns) 4 Fluorescence lifetime imaging is a 6 way to image FRET Fluorescence decays recorded with YC3.1 cameleon dissolved in buffer. Excitation at 420 nm excites the ECFP part. (Habuchi et al. Biophys J, 2002) 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% A family of Ca2+-sensitive switches and buffers helix-loophelix makes E-F hand x x x x Calmodulin MW ~ 17 kDa KCa ~ 14 mM for free calmodulin Calmodulin (CaM) : An abundant 149 amino acid, highly conserved cytoplasmic 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 complexes with many proteins, imparting Ca2+-dependence to their activities. Calmodulin folds around a target helix 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.) Design of CaMeleons: Expressible proteins for Ca detection 440 nm Low calcium: No FRET 480 nm YFP C N CaM MLCK CFP C 440 nm CFP High calcium: FRET N FRET YFP 535 nm 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) Ca-sensitive cameleon emission spectra Note two peaks no Ca emission intensity Ca YC3.1 cameleon more FRET Emission wavelength (nm) (Miyawaki, Roger Tsien et al., 1997) Cameleon emission combines two spectra EYFP ECFP emission intensity Ca no Ca YC3.1 cameleon emission ECFP EYFP There is FRET even with no Ca2+! Amount of FRET gives distance changes. It is not a large change. Ca-sensitive FRET reporter. How do calciums bind? (Miyawaki et al., 1997) green cameleon 1 fluorescence ratios 510/445 nm emission ratio 1.0 E104 C lower affinity E31 N higher affinity GC1 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'). 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, Miyawaki et al. 1999 paper Dynamic and quantitative Ca2+ measurements using improved cameleons Each 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 McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin Fig 1 Y66 G67 0.1 0.0 2.1 2 2.1 Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin 2 Fig 2AB YC2.1 2.1 3.1 Emission wavelength (nm) 2.1 3.1 Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin Fig 2CD 2.1 3.1 Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin 2.1 3.1 Fig 3 YC2 Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin YC2.1 Fig 4 YC2.1 500 uM 150 uM 40 uM YC3.1 Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin Fig 1. Andrea McQuate Fig 2a,b. Jacob Baudin Fig 2c,d. Anastasiia Stratiievska Fig 3. Benjamin Drum Fig 4. Jesse Macadangdang Fig 5. Jerome Cattin Fig 5 split 2.1 YC3.1 +- CaM Emission wavelength (nm)