Calcium Signals in Biological Systems Lecture 3 (2/9/0) Measuring intracellular Ca 2+ signals II: Genetically encoded Ca 2+ sensors Henry M. Colecraft, Ph.D. Challenges to measuring intracellular Ca 2+ 1. Diversity of Ca 2+ signals Interesting Ca 2+ signals span wide temporal and spatial ranges. 2. Selectivity of indicator for Ca 2+ Cells have mm concentrations of Mg 2+ vs. nm-µm for Ca 2+. 3. Delivery of indicator to cells Plasma membrane poses a formidable barrier 4. Fidelity of reporter 5. Targetability to distinct intracellular sites heterogenous distribution of [Ca 2+ ] in organelles and cytosol. Calmodulin: nature s Ca 2+ sensor Calmodulin is the major Ca 2+ binding protein in the cytosol. Structurally, has two lobes with four EF hands that bind Ca 2+. In Ca2+ free state (apocalmodulin), molecule is stretched out. Upon binding Ca 2+, molecule undergoes conformational change to activate downstream effectors.
Ca 2+ - and substrate-dependent conformational changes in calmodulin Green fluorescent protein (GFP) Aequoria Victoria Conformational change in calmodulin upon binding Ca 2+ and substrate brings the N- and C- terminal lobes in closer proximity compared to apocalmodulin. GFP crystal structure Expression of GFP in mammalian cells Expression of GFP in other organisms results in fluorescence. Hence, the gene encodes all the information required for posttranslational synthesis of the chromophore. GFP is widely used as a reporter gene to monitor gene expression. In-frame fusion of the gene for GFP to that of other proteins permits tracking of the subcellular localization of proteins. GFP β 2a -GFP β 4 -GFP Mutagenesis of GFP Several mutations have been engineered into wild-type GFP to maximize use in mammalian cells. F99S, M153T, V13A improved folding of the molecule at 37 C. Altering the sequence of GFP to reflect mammalian codon usage and use of strong promoters permitted improved expression levels in mammalian cells. Importantly, targeted mutations permitted the generation of fluorescent proteins with unique spectral properties.
Fluorescence spectra of XFP proteins A. Jablonski diagram Fluorescence B. Excitation and emission spectra Distinct XFPs have different brightness levels and unique excitation and emission spectra. Spectral overlap between emission and excitation spectra of specific XFP pairs permits design of fluorescence resonance energy transfer (FRET)-based assays. Fluorescence resonance energy transfer (FRET) FRET occurs when the following two conditions are met: (1) The emission spectrum of a fluorophore, termed the donor (D), overlaps with the excitation spectrum of a second fluorophore, termed the acceptor (A). (2) D and A are in close proximity. FRET does not involve emission and absorption of photons, but reflects the exchange of energy between two oscillating dipoles with similar resonance frequency. Theory of FRET The extent of energy transfer is a function of the distance between donor and acceptor (r) and the spectral overlap between them. The spectral overlap is usually described in terms of the Förster distance, R o. The rate of energy transfer (k T ) is given by, k T 1 R = τ r D where τ D is the lifetime of the donor in the absence of acceptor. The efficiency of energy transfer for a single donor-acceptor pair is given by, E = R Ro o + When r = R o, the rate of energy transfer equals the rate of emission of the donor, and efficiency of energy transfer is 0.5. R o is typically on the order of the size of biological macromolecules (30 0 Å), making FRET a powerful technique for measuring distances between proteins. o r
Strategy for cameleons Construct a gene encoding calmodulin and M13 sandwiched by donor and acceptor XFPs. In Ca 2+ -free state, CaM and M13 are disordered and stretched out, and XFPs are relatively far apart. Exciting the donor results in mostly donor emission. Upon binding Ca 2+, CaM binds to M13 decreasing the distance between donor and acceptor XFPs, thereby increasing FRET between them. Now, exciting the donor results in emission mostly from the acceptor XFP. Design of cameleons for bacterial expression Properties of cameleons in vitro Cameleon-1 consists CaM and M13 sandwiched by BFP and GFP. Protein expressed and purified from bacteria and spectral properties examined in vitro. Emission spectra of cameleon-1 measured following excitation at 380 nm. Two-humped emission spectra at 0 Ca 2+ significant resting FRET between BFP and GFP in the intact molecule. Polyhistidine tag permits column purification of protein from bacteria. Optimized splice sequences for different genes found by trial and error. CaM linked to M13 by glycine linker. Increasing Ca2+ resulted in increase in emission fluorescence intensity at 510 nm, and a decrease at 445 nm, i.e. increased FRET.
Ca 2+ titration curves of cameleon-1 Emission ratio change of cameleon-1 showed complicated dependence on Ca 2+ concentration. [ Ca 2+ ] = K ' d R R min Rmax R 1 n where, K d is the apparent dissociation constant, R max and R min are emission ratios in 0 and saturating Ca 2+, respectively, and n is the Hill coefficient. For wild-type cameleon-1 (open circles) the titration curve is biphasic with two apparent dissociation constants of 70 nm and 11 µm, and Hill coefficients of 1 and 1.8, respectively. Ca 2+ titration curves of cameleon-1 Mutation E104Q markedly reduces Ca 2+ affinity of the third Ca 2+ -binding loop of calmodulin. Applying this mutation to cameleon-1 (solid circles) eliminates the high affinity component of the wild-type response (K d = 4.4 µm; n = 0.7). Mutating the first Ca 2+ binding loop (E31Q; open triangles) further decreased the affinity of the low-affinity component of cameleon-1, while having no effect on on the high-affinity component (K d = 83 nm and 700 µm; n = 1.5 and 0.87). Mutations offer opportunities to engineer cameleons with different affinities extending extending the range of Ca 2+ concentrations that can be measured. Kinetic properties of cameleon-1 (E104Q) Improved properties of cameleon-2 Stopped-flow measurements of acceptor emission after rapid mixing with Ca 2+ revealed exponential relaxation. 1 2 + k obs = = ( kon[ Ca ] + k τ Calculated K d is on the same order as obtained from fits to the titration curve. Relatively slow kinetics make cameleon-1 inappropriate for tracking rapidly changing Ca 2+ concentrations. off ) Cameleon-1 was relatively ineffective as a reporter of [Ca 2+ ] inside mammalian cells because of the dimness of BFP. Second generation cameleon, yellow cameleon-2 was generated to resolve this issue. Yellow cameleon-2 contains ECFP and EYFP as donor and acceptor fluorophores, respectively. Emission spectra of yellow cameleon-2 in the absence and presence of Ca 2+ are shown (excited at 432 nm).
Design of cameleons for cellular expression Cameleon-reported Ca 2+ changes in HeLa cells Yellow cameleon-2 (74 kda) expressed in HeLa cells is present throughout the cytosol and excluded from the nucleus. Application of histamine induced oscillatory increases in emission ratio, indicating oscillations in [Ca 2+ ] cyt, which ultimately desensitized. ATP induced a reversible increase in [Ca 2+ ] cyt. R max determined by elevating extracellular Ca 2+ to 20 mm in the presence of Ca 2+ ionophore, ionomycin. R min determined by clamping extracellular Ca 2+ to 0 with EGTA, and chelating intracellular Ca 2+ with BAPTA-AM. Targeting cameleons to intracellular organelles: nucleus Targeting cameleons to intracellular organelles: ER/SR Cameleons containing a nuclear localization signal target specifically to the nucleus when expressed in HeLa cells. Unique advantage of genetically encoded Ca 2+ sensors is this ability to potentially target any subcellular location. Cameleons containing ER targeting and retrieval signals, are targeted to the ER when expressed in HELA cells. Resting emission ratio is high, reflecting the high Ca 2+ concentration in the ER. Stimuli which increase cytosolic [Ca 2+ ] (e.g. histamine and ATP) decrease ER Ca 2+, demonstrating the ER as the source of Ca 2+ signals in these paradigms. Resting emission ratio is identical to R max. This indicates that yellow cameleon-3er is saturated at rest. Hence, estimates of ER Ca 2+ concentration using this cameleon are unreliable.
Measuring ER/SR Ca 2+ with cameleon-4er GFP-based detection of protein association and dissociation Lower affinity yellow cameleon-4er is not saturated at resting ER [Ca 2+ ]. ER [Ca 2+ ] concentration estimated as 0 400 µm. Expression of yellow cameleon-2 as two separate pieces resulted in distribution of both proteins in the cytosol and nucleus. Stimuli that caused changes in Ca 2+ increased emission ratio in both the cytosolic and nuclear compartments. Hence, GFP-based FRET can be used to monitor protein-protein interactions in cells.