Due to the importance of Ca2+ in biological systems, the attempt to measure [Ca2+] has contin-‐
ued since 1920. Currently, numerous techniques or methods have been developed for analyzing cellular or subcellular Ca2+ activity. Currently used calcium sensors are mainly divided into two classes: chemical
fluorescent indicators generated by organic synthesis based on BAPTA [1,2-‐bis(o-‐aminophenoxy)ethane-‐ N,N,N',N'-‐tetraacetic acid] and bioluminescent calcium indicators containing photoproteins (Tsien et al., 1985, McCombs et al., 2008). BAPTA is the first organically synthesized small molecule designed on the basis of EGTA (ethylene glycol tetraacetic acid), which exhibits high metal selectivity for Ca2+ (Tsien,
1980). BAPTA became the essential component of several calcium dyes such as Indo 1, Fluo 3 and Ore-‐ gon green BAPTA (Grynkiewicz et al., 1985, Minta et al., 1989). The chemical fluorescent indicators can be excited at different wavelength ranges: UV wavelength excitation, such as Indo 1, Fura 2 and their derivatives (Grynkiewicz et al., 1985, Naraghi, 1997, Etter et al., 1994, Etter et al., 1996), and visible wavelength excitation, such as Fluo 3, calcium green, dextran conjugates (Grynkiewicz et al., 1985, Minta et al., 1989). Even though the UV-‐excitable Ca2+ indicators are still used due to their quantitative
ratiometric property, they are known to be more cytotoxic (Brakenhoff et al., 1996). Therefore, visible wavelength Ca2+ sensors have many advantages over UV-‐based Ca2+ indicators like less cytotoxicity, the
ability to monitor change of [Ca2+] with UV-‐sensitive compounds and emitting light within the region of
the electromagnetic spectrum with less severe background scattering (Takahashi et al., 1999). The ma-‐ jority of these indicators can produce greater dynamic range of signals like fluo 3 that can undergo 40-‐ 200 fold increase in fluorescence upon binding Ca2+ (Minta et al., 1989, Harkins et al., 1993). However,
the big disadvantage of these synthesized Ca2+ sensors is their non-‐specific targeting of membrane and
easy leakage from cell (McCombs et al., 2008). In addition, Ca2+ in denser or thicker tissues cannot be
detected due to the difficulty in loading these Ca2+ dyes and also, the emitted light signal is attenuated
Bioluminescent Ca2+ indicators mainly include two types of Ca2+ sensors: Ca2+-‐binding photopro-‐
teins and GFP-‐based indicators. Ca2+-‐binding photoproteins, such as aequorin and obelin etc, offer sim-‐
plicity for instrumentation by emitting visible bioluminescence through an intramolecular reaction upon binding Ca2+ (Shimomura, 1984, Campbell, 1974). Aequorin is the most widely used bioluminescent Ca2+
sensor which is composed of three parts: apoaequorin protein of molecular mass of 21 kDa, the lumino-‐ phore coeleterazine and molecular oxygen (Inouye et al., 1985, Inouye et al., 1989, Shimomura et al., 1988). The aequorin contains three Ca2+-‐binding sites and produces emission with Ca2+ binding to at
least two sites (Shimomura et al., 1963). Upon binding Ca2+ ions, the molecular oxygen in aequorin is
released and the luminophore coeleterazine is oxidized to coelenteramide, emitting blue light (465 nm) which increases as [Ca2+] increases between 10-‐7 and 10-‐4 M (Shimomura et al., 1963). However, its in-‐
ability to get into organelles limits the distribution of aequorin in cell (Shimomura et al., 1962). There-‐ fore, recombinant aequorins have become quite useful probes for calcium due to their ability to enter into various kinds of organelles without interfering with the physiological condition (Rizzuto et al., 1992). Obelin is a Ca2+-‐activated photoprotein that binds at least three molecules of Ca2+ to emit biolumines-‐
cence (Campbell, 1974). Compared to aequorin, obelin has faster onset of the bioluminescence in re-‐ sponse to binding Ca2+ (3 ms by obelin vs. 10 ms by aequorin) but less [Ca2+] sensitivity (Moisescu et al.,
1975). The GFP-‐based Ca2+ biosensor is a promising indicator that exhibits high specificity for location
and provides high accuracy for the measurement of subcellular Ca2+ signaling (Inouye et al., 1994). The
basic strategy for the design of GFP-‐based Ca2+ biosensor is the insertion or graft of a Ca2+ sensitive pro-‐
tein such as calmodulin, or Ca2+ binding proteins like troponin C, into different mutated types of GFP to
create various kinds of Ca2+ indicators. Therefore, fluorescence changes provide the information about
[Ca2+], which is produced by direct alteration in GFP or an increase in fluorescence resonance energy
transfer (FRET). Cameleons, as one kind of the successfully synthesized Ca2+ indicators, consist of two
with M13, which is a calmodulin-‐binding peptide with 26-‐residues from myosin light-‐chain kinase (Miyawaki et al., 1997). The calmodulin-‐M13 complex linked the two GFP mutants. When Ca2+ binds to
calmodulin, it induces the conformational change of the complex, resulting in decreased distance be-‐ tween the two GPF mutants accompanied by an increase in FRET. Two combinations of donor and ac-‐ ceptor GFP mutants have been designed: blue fluorescent protein (BFP)-‐GFP and cyan fluorescent pro-‐ tein (CFP)-‐yellow fluorescent protein (YFP) (Tsien, 1998). Troponin C is another Ca2+ binding protein
functioning only in muscle contraction (Heim et al., 2004). Troponin C is inserted between CFP and ci-‐ trine that is a yellow fluorescent protein derivative, and this forms troponeons that is another type of FRET-‐based Ca2+ indicators. Compared to cameleons, troponeons exhibit better performance in target-‐
ing to a specific subcellular domains (Takahashi et al., 1999). GFP-‐based Ca2+ indicators have been widely
used for its many advantages over the chemical fluorescent probes, such as the ratiometric measure-‐ ment of [Ca2+], brighter fluorescence, high sensitivity, precise expression in targeted intracellular com-‐
partments and so forth, although they demonstrated fewer changes than most chemical fluorescent Ca2+ indicators and pH-‐sensitivity seen in some GFP variants as the disadvantages (Takahashi et al., 1999,
Kneen et al., 1998).
Yang’s lab has been devoted to exploring site-‐specific Ca2+ binding affinity of designed calcium
sensors (Figure 1.15). They have successfully created a Ca2+ sensor by grafting an EF-‐hand motif with
Ca2+ binding site into EGFP (enhanced green fluorescent protein) that contains F64L and S65T (Zou et al.,
2007). The grafted Ca2+ sensor (G1) emitted a dual 510 nm fluorescence intensity ratio metric change
when excited at 398 nm and 490 nm wavelengths. The fluorescent emission ratio (measured at 510 nm) for 385 nm to 480 nm excitation is indicative of [Ca2+]. However, the dynamic range is small with only
10-‐15% change observed in mammalian cell imaging (Zou et al., 2007). The hypothesis for the design of EGFP-‐based Ca2+ sensor is that alteration of chromophore is associated with the conformational change
EGFP and site directed mutations at that area were designed to bind small molecules such as the GFP-‐ based zinc sensors (Kikuchi et al., 2004). Therefore, a new Ca2+ binding pocket was designed via site di-‐
rected mutagenesis on the surface of EGFP. A series of different combinations of amino acid substitu-‐ tions with negatively charged side chains that can coordinate with Ca2+ was designed to (Tang et al.,
2011). This de novo design of Ca2+ sensors can provide theoretical support for developing GFP-‐based
biosensors for diverse molecules by the means of site-‐directed mutagenesis.
Figure 1.16 Scheme of EGFP-‐based Ca2+ biosensor. EGFP is used as a scaffold protein and EF-‐hand III of calmodulin was grafted into the fluorescent sensitive location of EGFP to produce Ca-‐G1; negatively charged amino acids were introduced on the surface of three antiparallel beta sheets of EGFP forming a pentagon to bind Ca2+.