Patent Application
20130231262
The present invention generally relates to engineered proteins. More particularly, this invention relates to an engineered protein for calcium handling within human cells, the determination of SERCA activity for optimization of cardiac function, and the potential for pharmacological treatment.
The sarco(endo)plasmic reticulum calcium adenosine triphosphatase (SERCA) is the ion-motive ATPase responsible for maintaining the 7,000 fold Ca gradient across the membrane of the endoplasmic reticulum (ER). Specifically, SERCA is a P-type ion pump responsible for transporting Ca ions from the cytoplasm to sarcoendoplasmic reticulum (SR) in muscle cells.
SERCA plays a particularly important role in striated muscle, where Ca release and reuptake determines the contraction and relaxation of the muscle. This process is critical during cardiac diastole (relaxation), as the muscle must relax to allow for adequate filling of the ventricles with blood. SERCA function is also important for systole (cardiac contraction), as it is solely responsible for creating the large store of SR Ca that is released to initiate activation of the muscle.
In particular, optimal calcium handling is critical for normal cardiac function; deranged SERCA activity has been implicated as a cause and an effect of heart failure. This and other P-type ATPases are considered high value targets for pharmacological treatment of heart failure. Prior investigations into SERCA have used optical methods to study the dynamic transitions of SERCA protein structure. These studies generally used extrinsic fluorescent probes.
Traditional fluorescent labeling schemes require thiol- or amine-reactive chemistries, some of which are known to inactivate ATPases. For example, a known hybrid approach utilizes a CFP-SERCA labeled with a fluorescein dye. All such assays require detergent solubilization and purification of proteins, and reconstitution into artificial membranes. Such steps risk compromising protein function. A significant disadvantage associated with these conventional methods is that they are not compatible with live cell experiments.
In view of the above, it can be appreciated that it would be desirable if improved methods were available for studying SERCA that were capable of being used in vivo and do not require protein purification or chemical labeling of SERCA, or reconstitution into artificial membranes.
The present invention provides a method and engineered proteins for use therewith suitable for studying SERCA that are capable of being used in vivo and do not require protein purification or chemical labeling of SERCA, or reconstitution into artificial membranes.
According to a first aspect of the invention, an engineered protein for calcium handling within human cells includes a two-color SERCA construct comprising three component proteins fused together. The three component proteins comprise a blue fluorescent protein (cerulean donor), SERCA2a and a yellow fluorescent protein (YFP acceptor), or a red fluorescent protein (tagRFP acceptor), SERCA and a green fluorescent protein (GFP donor).
According to a second aspect of the invention, a method of determining SERCA activity for optimization of cardiac function includes resolving structure changes of a two-color SERCA construct comprising three component proteins fused together. The two-color SERCA construct is catalytically active and able to pump calcium following the step of resolving structure changes.
A technical effect of the invention is the ability to study the function and structure of SERCA in vivo. In particular, it is believed that, by forming two-color SERCA constructs comprising fluorescent proteins, the engineered proteins will remain catalytically active and able to pump calcium following the step of resolving structure changes, and therefore SERCA can be studied during its normal function.
Other aspects and advantages of this invention will be better appreciated from the following detailed description.
The present invention is generally applicable to methods and assays for studying the structure and function of SERCA. SERCA is believed to undergo conformational changes during catalytic cycling. In particular, X-ray crystallography have provided many atomic resolution SERCA structures suggesting that the relative motions of SERCA cytoplasmic domains result in opening and closing of the cytoplasmic headpiece during the transition between the E1 (Ca-bound) and E2 (Ca-free) enzymatic substrate (
The E1 (Ca bound) and E2 (Ca free) conformations are characterized by different relative positions of three cytoplasmic domains (N, A, P), collectively known as the cytoplasmic headpiece. Conformational changes in the headpiece are transmitted to the trans-membrane (TM) domain to alter the occlusion and affinity of the Ca binding sites. The first X-ray crystal structures of SERCA suggested that Ca induces a large increase in the separation of the N- and A-domains, but recent crystallography and in vitro fluorescence resonance energy transfer (FRET) experiments have depicted a closed cytoplasmic headpiece for Ca-bound SERCA, and suggest that the conformational change may be smaller than previously thought.
To directly quantify SERCA headpiece conformational changes in live cells, the present invention provides methods of measuring intramolecular fluorescence resonance energy transfer (FRET) between fluorescent protein fusion tags directed to various sites on the SERCA cytoplasmic domains. The methods utilize engineered SERCA proteins to determine how the domain separation distance changes with Ca binding. Unlike prior methods of studying SERCA, the methods described herein takes advantage of the intrasequence tag, which can be fused to sites on SERCA. In addition, the fluorescent construct is entirely biosynthetic and compatible with live cell measurements. Therefore, unlike prior methods, the structure and function of SERCA can be studied in vivo. This is important since many drug candidates identified from in vitro screens later fail because they cannot penetrate cell membranes or because they are toxic to cells. This is not identified in the first screen, only in subsequent follow-up permeability/toxicity screens in cells or in live animals. This may add significantly to the cost of screening. 2-color SERCA avoids this by combining the primary optical screen together with a live cell screen, all together in a single step. By quantifying relative changes in probe separation distance, it is believed that insight can be gained into the conformational changes of the SERCA cytoplasmic headpiece.
According to an aspect of the invention, engineered proteins were produced to report changes in the conformation of the SERCA. The proteins are a two-color SERCA construct comprising three component proteins fused together. Examples of the three component proteins include a blue fluorescent protein (cerulean donor), SERCA2a and a yellow fluorescent protein (YFP acceptor), or a red fluorescent protein (tagRFP acceptor), SERCA and a green fluorescent protein (GFP donor). Although the invention will be discussed hereinafter in reference to the above two examples, additional constructs are foreseeable. In addition, the methods described herein may be applicable to other embodiments, for example, constructs could be produced for the highly homologous sodium/potassium ATPase (NKA, particularly with regard to the cardiac environment), an ABC transporter (useful for screening drugs for cystic fibrosis, cancer, multi-drug resistance), skeletal muscle SERCA (useful for screening drugs for treatment of muscular dystrophy), or other SERCA species (useful for screening drugs for treatment of malaria). Each construct is self-contained, encoded by a single plasmid.
In a first set of experiments studying SERCA, mCerulean (Cer) was fused to the N-terminus of canine SERCA2a to form Cer-SERCA-YFP constructs. This fusion position is in the A-domain of the pump, represented in
In producing the constructs, regions important for SERCA function were avoided. Fluorescence microscopy of AAV-293 cells (Agilent, Santa Clara, Calif.) expressing 2-color SERCA was performed in glass-bottom chambered coverslips (Matek Corporation, Ashland, Mass.) coated with poly-D-lysine (Sigma-Aldrich) 18-24 hours post-transfection with MBS mammalian transfection kit (Stratagene, La-Jolla, Calif.).
To determine the effect of Ca on the SERCA structure, the cells were permeabilized for 1 minute in 50 mM ionomycin in growth medium (90% Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, 1% glutamine). To reduce intracellular Ca, the cells were permeabilized with 50 μM ionomycin in 5 mM EGTA in phosphate-buffered saline or relaxing solution (100 mM KCl, 5 mM EGTA, 5 mM MgCl2, 3.2 mM ATP, 10 mM imidazole pH=7.2). Confocal microscopy was performed using a Leica SP5 confocal microscope equipped with a 1.3 N.A. 63× water immersion objective. Excitation was accomplished with Ar laser illumination at 458 nm for Cer, 514 nm for YFP and FM 4-64, with emission bands of 467-512 nm for Cer, 528-565 for YFP, and 674-797 nm for FM 4-64, using sequential image acquisition. Total internal reflection fluorescence (TIRF) microscopy was performed with a Nikon inverted microscope (Ti-e) equipped with a 100× oil-immersion objective with (N.A.=1.49) and a cooled CCD camera (Coolsnap K4, Photometrics, Tucson, Ariz.). Through-objective TIRF excitation was achieved with a 449 nm diode laser (for Cer) or 514 nm Ar laser (for YFP). The laser incident angle was adjusted to create an evanescent field that illuminated the plasma membrane in contact with the surface substrate and a thin section of endoplasmic reticulum. Widefield lamp excitation was used for FRET measurements, using computer-controlled filter wheels, as known in the art. Fluorescent images were recorded with a cooled EM-CCD camera (Andor Ixon, Belfast, Northern Ireland). The RG-SERCA constructs in the second set of experiments discussed below were produced by substantially similar methods as the YFP constructs discussed above.
To test whether the Cer-SERCA-YFP constructs show structural substrate-dependent FRET, the steady state was measured in the presence and absence of thapsigargin and Calcium.
To evaluate Ca transport activity for 2-color SERCA constructs, a live-cell Ca uptake assay was performed. Heterogeneously transfected populations of AAV-293 cells were incubated with cell permeant Ca indicator dye X-rhod 1 (AM) (Invitrogen, Carlsbad, Calif.). Transfected and untransfected cells were distinguished on the basis of the intensity of YFP fluorescence emission. Release of Ca from intracellular stores was accomplished by stimulating purinergic receptors with extracellular application of 100 μM ATP. Accumulation of Ca in the cytosol was quantified as an increase in X-rhod 1 fluorescence, and was the net result of Ca release counterbalanced by Ca extrusion and uptake processes, including SERCA activity. Exogenous SERCA activity was detected as a decrease in ATP-stimulated cytosolic Ca accumulation relative to untransfected cells in the same microscopic field. Three minutes after application of extracellular ATP, cells were treated with 10 μM Tg to determine the size of the Ca store remaining in the ER. The magnitudes of the ATP- and Tg dependent Ca transients were quantified by integrating the area under the trace of X-rhod 1 fluorescence vs. time. The integrated area of Ca transients in 2-color SERCA transfected cells was compared to corresponding control (untransfected cells) using a paired t-test, with a p-value of greater than 0.05 taken to indicate a significant difference. To determine whether the 2-color SERCA samples were regulated by phospholamban (PLB), cells transfected with 2-color SERCA were compared to separate samples transfected with both 2-color SERCA and YFP-PLB using an unpaired t-test, with a p-value of greater than 0.05 taken to be a significant difference. For Cer-SERCA2a and construct 509, enzymatic activity was also quantified in cell homogenates by spectrophotometric measurement of the rate of NADH consumption in an enzyme-coupled activity assay. Conventional cell transfection did not yield adequate expression of SERCA protein for this assay, so AAV-293 cells were infected with adenoviruses encoding Cer-SERCA2a or 509 resulting in much greater protein expression, as quantified by immunoblotting.
FRET was quantified using the E-FRET (3-cube) method. FRET efficiency (E) was calculated according to the relationship
E=IDA−aIAA−d(IDD)IDA−aIAA+G−d(IDD)
where IDD is the intensity of fluorescence emission detected in the donor channel (472/30 nm) with excitation of 427/10 nm; IAA is acceptor channel (542/27 nm) emission with excitation of 504/12 nm; IDA is the “FRET” channel, with 542/27 nm emission and excitation of 427/10 nm; a and d are cross-talk coefficients determined from acceptor-only or donor-only samples, respectively. Values of d=0.7 (for Cer) and a=0.074 (for YFP) were obtained. G is the ratio of the sensitized emission to the corresponding amount of donor recovery, which was 3.2 for this set of experiments. Probe separation distance (R) was calculated from the relationship described by Förster, R=(R0)[(1/E)−1)1/6, where E is the measured FRET value and R0 is the Förster radius, which is 49.8 Å for the Cer-YFP pair. E-FRET measurements were also compared with FRET obtained by the photobleaching method. All error bars represent mean±SE.
To verify that the fluorescent protein fusion tags did not disrupt SERCA catalytic function, Ca uptake was measured for all constructs using a live-cell Ca uptake assay.
Pump activity was also evaluated for some constructs using an enzyme-linked ATPase assay performed on cell homogenates.
All of the constructs showed fluorescence localization patterns consistent with localization in the endoplasmic reticulum. TIRF microscopy of cells expressing construct 509 showed that Cer and YFP signals were highly colocalized in a reticulated pattern (
The observed Ca transport and ATPase activity (
The average FRET observed for 2-color SERCA was found to depend on the YFP insertion site and the enzymatic substate of the pump (
Additional characterization of construct 509 showed that the time course of the Tg-dependent FRET change was dependent on the concentration of Tg applied to the cells (
Based on the above described tests, it is believed that SERCA intramolecular FRET is increased by Ca binding for all four 2-color SERCA constructs. This suggests a decrease in the distance between the Cer donor fluorophore and the YFP acceptor, which implies that the SERCA cytoplasmic headpiece becomes more compact after Ca binds to the SERCA transmembrane domain. The apparent donor-acceptor separation distances are summarized in Table 1. Several uncertainties apply to these values. Distances were calculated using the assumption that the average relative dipolar orientation of the probes was random (κ2=2/3). Nonspecific FRET was not subtracted from the measured FRET values, because there was no evidence of a concentration-dependent increase in FRET (
Compared to EGTA, the apparent probe separation distance decreased with high Ca by approximately 23% (from 80 Å to 62 Å) for the most responsive construct, 509 (Table 1). This change was somewhat smaller in magnitude than the 30+Å change predicted by the first X-ray crystallographic structures (
Supporting the hypothesis of large-amplitude headpiece transitions is a recent study using fast scanning atomic force microscopy, which demonstrated 23 Å changes in the height of the SERCA headpiece relative to the surface of the bilayer during catalytic cycling. The data are also consistent with molecular dynamics simulations performed on SERCA starting at the open conformation of SERCA obtained in the first crystal structure (
Of the YFP constructs described here, the least responsive was 661, which showed low initial FRET efficiency and small changes with Ca or Tg. This is consistent with inefficient maturation of the YFP tag (
It was shown that steady-state FRET experiments can resolve structure changes of the Cer-SERCA-YFP constructs. The data suggest N-domain sites got farther to the N-term donor after TG, while P-domain and the C-terminus got slightly closer. For all labeling sites, FRET increased with Calcium, suggesting the pump assumes a more compact conformation with Ca-binding. Preliminary data suggest that the pump is still catalytically active, and able to pump calcium.
In a second set of experiments, additional testing was performed with an another 2-color SERCA, RG-SERCA, that is believed to benefit from a higher efficiency FRET pair (
In the second set of experiments, the 509 construct was again utilized as well as a RG-SERCA construct. The Cer-YFP pair had a Förster distance (R0) of 49.8 Å and the GFP-tagRFP pair had an R0 of 58.3 Å. Expression of 2-color SERCA in AAV-293 cells was performed as previously described in reference to the first set of experiments. Briefly, cells were transfected with plasmids encoding 2-color SERCA using the MBS mammalian transfection kit (Stratagene). The transfected cells were trypsinized and re-plated onto poly-D-lysinecoated glass bottom dishes and allowed to attach for 2-4 hours before being used for measurements. Cotranfection of 2-color SERCA with non-fluorescent S16A-PLB or S16E-PLB was performed at a plasmid molar ratio of 1:10. For single molecule experiments, the protein was solubilized in detergent as follows: 0.1% dodecylphosphocholine (DPC) (Sigma Aldrich) in phosphate buffered saline was gently layered over AAV-293 cells expressing 2-color SERCA, and incubated at room temperature for 45 minutes. The solution was withdrawn and centrifuged at 16,000 g for 10 min, and the supernatant was transferred to Matek chambered coverglass for spectroscopy.
Adenoviral vectors of 2-color SERCA were prepared using AdEasy system (Stratgene, La Jolla, Calif.). Adult cardiac ventricular myocytes were enzymatically isolated from adult New Zealand White rabbits. Myocytes were transferred to culture vessels and washed with fresh PC-1 medium (Lonza, Basel, Switzerland). 2-color SERCA adenoviruses were added at a multiplicity of infection of 1000. Cells were paced for 48 hours in culture using a C-Pace EP pacer (IonOptix, Milton, Mass.) set to 10 volts with a frequency of 0.1 Hz and 5-ms pulse duration. During spectroscopy experiments, electrical pacing of cardiac myocytes was performed with a stimulator (Grass S44, Astro-Med, Inc., West Warwick, R.I.), with 50 V stimulation, 5 ms duration, 0.25 Hz.
Time correlated single-photon counting (TCSPC) and fluorescence imaging were performed on an inverted confocal microscope (TCS-SP5, Leica Microsystems) equipped with a 63×1.20 NA water immersion objective (Leica Microsystems) and a pulsed Ti-Sapphire laser (Coherent Inc.), with excitation at 840 nm. Emission was split by a dichroic filter centered at 560 nm and passed through filters of 500-550 nm (for EGFP fluorescence) and 607-683 nm (for Tag-RFP fluorescence). Avalanche photo diodes (SPCM-AQRH, Perkin Elmer) were used for detection of photons. The signals from the photo diodes passed to a pulse inverter (APPI-D, Becker & Hickl GmbH) and 20-dB attenuator and a TCSPC router (HRT-41, Becker & Hickl GmbH) for simultaneous data collection of both emission channels. The signal output and the routing information from the router were transmitted to a TCSPC card (SPC-830, Becker & Hickl GmbH) to record temporal information for each detected photon. A synchronization reference signal for the TCSPC measurements was obtained by directing a portion of the excitation laser onto a photo diode (PHD-400-N, Becker & Hickl GmbH). Temporal information was recorded as two different time tags for each detected photon: t1, the microscopic arrival time relative to the previously measured synchronization reference signal, and t2 indicating the macroscopic arrival time of the photon measured with respect to the start of the experiment. Time tag t1 provides information about ns scale fluorescence lifetime and t2 measures the timecourse of the experiment with ms time resolution. TCSPC data were analyzed with a sliding-scale method to obtain fluorescence lifetime (τ) histograms from recorded photon arrival times. Briefly, the train of photons detected in the FRET donor channel was analyzed in overlapping blocks of 200 consecutive photons, with τ determined for each block by maximum likelihood estimation (MLE). A histogram of the t1 arrival times of the photons was used to calculate the likelihood function. The probability distribution for this histogram was approximated by a single exponential decay of the form e−t/τ. Maximizing the logarithm of the likelihood function for the single exponential decay yields
where ni indicate the number of photons in the ith bin of the histogram and
ω is the width of the time bins in the histogram and ti=iω with 0≦ti≦T. The root of this equation yields the MLE estimate of the τ value of the fluorophore for the corresponding t2 time bracket. The analysis was automated with a custom MatLab program. The method was validated by determining the fluorescence lifetime of Rhodamine 6G in water at room temperature. FRET efficiency was calculated from measured τ values according to the relationship (1−(τDA/τD)), where τDA values are measurements of 2-color SERCA fluorescence lifetime and τD is the average τ of the donor-alone control. FRET efficiency histograms were fit to a sum of four Gaussian peaks with Origin (OriginLab Corp., USA). All fit parameters (the center value, the width and area of each Gaussian) were iteratively varied until convergence was obtained. Parameter mean values were obtained from five independent measurements.
The fluorescent protein FRET pairs for Cer-SERCA-YFP and RG-SERCA have different R0, τD, and emission spectra. The constructs also have reciprocal donor/acceptor positions. Despite these differences, the distances calculated from FRET substates were very similar for Cer-SERCA-YFP and RG-SERCA (Table 3). The agreement of these values suggests that the probe separation distance estimate was not dominated by κ2 or beat-to-beat changes in pH or autofluorescence.
FRET efficiency trajectories calculated for each single molecule transit were obtained for analysis of SERCA structural dynamics. FRET efficiency values measured from overlapping blocks of 200 consecutive photons were assigned to specific conformational States I-IV identified from previous Gaussian fitting of FRET efficiency histograms (Supplemental Table 1). The full width at half maximum of each Gaussian distribution was used as a threshold for substate assignment. The duration of time spent in each FRET state before transition to another FRET state was quantified, and a histogram of observed dwell times was generated using a custom analysis routine in MATLAB. The histogram of the dwell times was fit by a biexponential function.
Single molecule FLDA experiments were performed with Cer-SERCA-YFP. A histogram of measured τ values revealed a large range of lifetimes from 1.2 ns to 2.9 ns (
To survey resolvable conformations of SERCA in vitro, pulsed excitation single molecule fluorescence spectroscopy was performed with fluorescence lifetime distribution analysis (FLDA) using RG-SERCA solubilized in dodecylphosphocholine (DPC).
Detergent-solubilized single molecules yielded time-trajectories of up to 800 ms in duration. These showed apparent stochastic transitions between high FRET and low FRET conformations (
To investigate the structural heterogeneity of SERCA in cardiac muscle, 2-color SERCA was expressed in cultured enzymatically isolated rabbit myocytes.
The effect of adrenergic stimulation on SERCA structural heterogeneity was also investigated. The effect of 8 agonist (isoproterenol) was not apparent during diastole, with the majority of SERCA assuming low FRET conformations (states I and II) (
Overall, experiments with electrically paced cardiac myocytes suggest that low FRET states (I, II) correspond to Ca-free (E2) conformations while high FRET states (III, IV) correspond to Ca-bound conformations. Consistent with this interpretation, Tg treatment abolished states III and IV, leaving SERCA approximately evenly divided between states I and II (
To directly test whether State IV represents a PLB-free SERCA structure or a unique phosphorylated PLBSERCA regulatory complex conformation, the distribution of RG-SERCA conformations was quantified in AAV-293 cells, which lack endogenous PLB. Confocal microscopy revealed the expected co-localization of the fluorescence of the GFP (
It is noteworthy that there is a 30 Å difference in distance between the most extreme open E2 (I) and closed E1 states (IV) (Table 3). This is a larger difference than would be predicted from X-ray crystal structures. Significant structures from recent X-ray crystallography studies show on average a of about six A difference in the distance between the FRET donor and acceptor fusion sites for E1 structures versus E2 structures. A significant outlier is the first E1 crystal structure 1SU4, characterized by a widely open headpiece structure. It has been proposed that this was an artifact arising from the absence of nucleotide in the crystallization conditions. While we did not directly detect an open E1 structure, the presence of a very low FRET E2 state with a 94 Å probe separation distance suggests that the 1SU4 structure represents a real conformation that is significantly populated in vivo (Table 4). It is believed that it is unlikely that the low FRET state arises from partially expressed proteins lacking the acceptor fluorophore, as the pump was observed to reversibly sample this state (
In addition to revealing the intrinsic FRET efficiency of discrete FRET states, Gaussian fitting also provided estimates of the variability of FRET observed for each state. These parameters are obtained from the peak center position and peak width, respectively. In general, histograms obtained from dilute single molecules in detergent solution were better resolved than those obtained from a higher density of fluorescent molecules in cell membranes. This is consistent with the expected ensemble averaging of multiple molecules diffusing simultaneously through the excitation volume in the cell. However, despite some loss of peak resolution in live cell experiments, it was still possible to observe an inverse relationship between Gaussian width and peak center position (
In view of the above, it is believed that the new FRET constructs were properly localized, functional, and responsive to conformational changes. Reversible transitions between four discrete conformations are consistent with fast (80 μs) Brownian motions and slow (690 μs) catalytic motions. These are large-amplitude transitions, producing an about 30 Å change in FRET pair separation distance. Low FRET states are consistent with open, dynamic structures, and prevail in the low Ca conditions that favor the SERCA E2 enzymatic substate. High Ca, which favors SERCA E1 substate, stabilizes high FRET states, with closed, rigidly ordered cytoplasmic headpiece conformations. The data are consistent with previous conclusion that the SERCA cytoplasmic headpiece closes with Ca-binding. Significantly, phosphorylation of phospholamban does not dissociate it from SERCA, nor does Ca-binding to SERCA abolish the regulatory complex. Instead, Ca-binding to SERCA and phospholamban phosphorylation together are believed to induce the SERCA cytoplasmic headpiece to sample a unique high FRET conformation. Taken together, the results predict several novel states that are not represented in the available X-ray crystal structures: an E2 state with a dynamically disordered open cytoplasmic headpiece (
The constructs are distinct from previous approaches in that they use only genetically encoded fluorophores. Thus, they do not require labeling with exogenous dyes. The plasmid DNA encoding the constructs are suitable for transfection into mammalian cells. The encoding DNA can be transfected into mammalian cells by standard methods, or stable cell lines can be created, making the supply of drug screening cells indefinitely expandable. The ligand-response of the constructs is large and easily resolved by steady-state fluorescence measurements of live cells. All of the constructs are adaptations of the SERCA protein thereby retaining SERCA's enzymatic functions and ability to bind the two major ligands tested above; calcium and the drug thapsigargin. Experiments show that the pumps are still catalytically functional. Three of the constructs exploit intrasequence labeling of the N- and P-domains.
Potential applications of the constructs include reporting of the structural state of the enzyme during normal function. Unlike prior methods, the engineered proteins can sense changes in the conformation of SERCA itself, rather than measuring changes in the interaction of SERCA and another protein. The principal intended use of these constructs was to perform fluorescence correlation spectroscopy experiments to observe rates of structure transitions under various physiological conditions. In addition, the constructs may be used as targeted calcium sensors. The probes may be useful for measuring localized release of calcium from the sarco(endo)plasmic reticulum with high spatial resolution. Further, the constructs may be used as fluorescent sensors for high-throughput screening of drug libraries.
While the invention has been described in terms of specific embodiments, it is apparent that other forms could be adopted by one skilled in the art. For example, the physical location of the intrasequence tag could differ from that shown, and materials and processes other than those noted could be used. Therefore, the scope of the invention is to be limited only by the following claims.
This application claims the benefit of U.S. Provisional Application No. 61/606,647, filed Mar. 5, 2012, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61606647 | Mar 2012 | US |
