Photolabile Chelator Research Articles

Light-controlled gene expression in yeast using photocaged Cu2+

The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.

Tuning caged calcium: Photolabile analogues of EGTA with improved optical and chelation properties

The physico-chemical properties of several Ca 2+-selective, photolabile chelators are described. These molecules have been developed as part of an effort to produce a caged Ca 2+ that improved upon the Ca 2+ chelation properties and light absorption capability of nitrophenyl-EGTA (NP-EGTA). Four dimethoxy- ortho-nitrophenyl derivatives of EGTA (called DMNPE-1 through -4), and one analogue of EGTA (DMNPE-5) have been characterized, each of which is bisected upon irradiation. One of these cages has a higher affinity than NP-EGTA: DMNPE-4 has a K d for Ca 2+ of 48 nm at pH 7.2 (19 nM at pH 7.4). Furthermore, this cage has a large extinction coefficient of 5120 M −1 cm −1 at 350 nm (cf. 975 M −1 cm −1 for NP-EGTA). The other physico-chemical properties of DMNPE-4 are: quantum yield of photolysis of 0.09; bipasic Ca 2+ release kinetics (70% released with a rate of about 48,000 s −1 and 30% at 1.5 s −1) and photoproducts that bind Ca 2+ with very low affinity ( K d in the range of 2 mM, pH 7.2), hence most of the bound Ca 2+ is released rapidly and efficiently upon photolysis. Thus, DMNPE-4 has a unique combination of properties that make it an extremely effective Ca 2+ cage.

Millisecond Time Resolved Photo-CIDNP NMR Reveals a Non-Native Folding Intermediate on the Ion-Induced Refolding Pathway of Bovine α-Lactalbumin.

Aspects of the structure of the intermediate populated after 200 ms in the Ca2+ -induced refolding of α-lactalbumin have been derived by time-resolved photo-CIDNP NMR methods. Refolding at constant denaturant concentration was initiated by laser-induced ion release from photolabile chelators. The NMR data demonstrated that part of the polypeptide chain in the β-domain of α-lactalbumin samples adopt non-native conformations while a hydrophobic core of the α-domain is already formed.

A new caged Ca 2+, azid-1, is far more photosensitive than nitrobenzyl-based chelators

Background: Photolabile chelators that release Ca 2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca 2+ affinity, selectivity for Ca 2+ over Mg 2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties. Results: Azid-1 is a novel caged calcium in which a fluorescent Ca 2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca 2+ with a dissociation constant (K d) of ∼230 nM, which changes to 120 μM after photolysis with ultraviolet light (330–380 nm). Mg 2+ binding is weak (8–9 mM K d) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40–170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N 2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-with-drawing ability of the amidoxime cation reduces the chelator's Ca 2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca 2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca 2+ transient in triggering synaptic plasticity. Conclusions: Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca 2+ in studies of the many Ca 2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.

Millisecond studies of calcium-dependent exocytosis in pituitary melanotrophs: comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen

DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca] i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (C m) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]; was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This ‘loading transient’ reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca] i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca] i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Cal+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187–191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca 2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93–104]. Although NPE is somewhat less effective than DMN in raising [Ca] i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

Using Caged Calcium to Study Sea Urchin Egg Cortical Granule Exocytosis in Vitro

We present a simple method for releasing calcium from the photolabile chelator DM-nitrophen in a microscope chamber and following the calcium-triggered exocytosis of sea urchin egg cortical granules. With this method, calcium can be introduced at different rates and the biological response can be monitored under these conditions. We find that this "uncaging" of calcium, but not magnesium, barium, or strontium (up to 500 μM), from DM-nitrophen triggers exocytosis. The timing and identity of which granules first fuse with the plasma membrane appear to be stochastic, although there does seem to be some subsequent positive cooperativity in the fusion of neighboring granules. Calcium-triggered exocytosis is a threshold phenomenon, and this threshold appears to be independent of the rate of calcium uncaging. Mild treatment with N-ethylmaleimide shifts this threshold so that higher concentrations of calcium are required for exocytosis and decreases the apparent rate of exocytosis despite the fact that all of the granules remain fusion competent, albeit with higher concentrations of calcium.

Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis.

The synthesis and properties of a caged calcium are described. The compound is an ortho-nitrophenyl derivative of EGTA. It is synthesized in 10 steps and with 24% overall yield. The photosensitive chelator, nitrophenyl-EGTA, has a Kd value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Upon exposure to UV radiation (approximately 350 nm), the chelator is cleaved, yielding iminodiacetic acid photoproducts with low Ca affinity (Kd = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence of Ca2+ is 0.20. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with nitrophenyl-EGTA-Ca2+ complex, in the presence of 1 mM free Mg2+, maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in 18 ms at 15 degrees C. Nitrophenyl-EGTA provides a tool for the investigation of the mechanism of Ca(2+)-dependent physiological processes, since under conditions of normal intracellular Ca2+ and Mg2+ concentrations, only Ca2+ is bound by the photolabile chelator and on illumination released rapidly and in high photochemical yield.

The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen

New procedures are described for producing brief transients and reversible elevations in [Ca] that can be used to quantitatively control the concentration of cytoplasmic calcium. If the photolabile calcium chelator DM-nitrophen, partially bound to calcium, is exposed to steady illumination, [Ca] can be raised from a few nM to up to 10 μM for durations of 100 ms or longer, depending on light intensity and duration. An association rate of calcium with nitrophen of 1.5 × 10 6 M −1s −1 was estimated from measurements of [Ca] using the fluorescent indicator Fluo-3, and calcium was found to speed the photolysis of nitrophen 2.5-times. Partial photolysis of DM-nitrophen partly loaded with calcium elicits a [Ca] spike of over 100 μM lasting about 1 ms, depending on intensity and duration of the light flash. Simulations of the reactions involved predict changes in Fluo-3 fluorescence measured at high time resolution with a laser scanning confocal microscope. These procedures have been applied in physiological experiments to generate cytoplasmic [Ca] spikes and pulses and study the cellular responses to them.

Activation and relaxation mechanisms in single muscle fibres.

The effect of Ca2+ on the time course of force generation in frog skinned muscle fibres has been investigated using laser flash photolysis of the caged-calcium, either nitr-5 or DM-Nitrophen. Gradations in the rate and extent of contraction could be achieved by changing the energy of the laser pulse, which varied the amount of caged Ca2+ photolysed and hence the amount of calcium released. The half-time for force development at 12 degrees C was noticeably calcium-sensitive when small amounts of calcium were released (low energy pulses) but did not change appreciably for calcium releases which produced a final tension of more than 50% of the maximal tension at pCa 4.5. This result is unlikely to be due to calcium binding to the regulatory sites of troponin C when on the thin filament, as this process is considered rapid (kon 10(8) M-1 s-1, koff 100 s-1). Our experimental results show that force develops relatively rapidly at intermediate Ca2+ which produce only partial activation (i.e. 50% Pmax or greater). This would not be the case if the affinity of the regulatory sites changes slowly with crossbridge attachment. The kinetics of calcium exchange with the regulatory sites may be much more rapid than crossbridge cycling, so that if calcium binding to a particular functional unit induces crossbridge attachment and force production, the force producing state may be maintained long after calcium has dissociated from that particular functional unit. The relaxation of skinned muscle fibres has also been successfully studied following the rapid uptake of Ca2+ by a photolabile chelator Diazo-2, a photolabile derivative of BAPTA, which is rapidly (> 2000 s-1) converted from a chelator of low Ca2+ affinity (Kd 2.2 microM) to a high affinity chelator (Kd 0.073 microM). We have used single skinned muscle fibres from both frog (actin regulated) and scallop striated muscle (myosin regulated), to study the time course of muscle relaxation. This procedure has enabled us to examine the effects of the intracellular metabolites, ADP, Pi and H+ upon the rate of relaxation.(ABSTRACT TRUNCATED AT 400 WORDS)

Rate of release of Ca2+ following laser photolysis of the DM-nitrophen-Ca2+ complex.

The determination of the rate of release of Ca2+ by pulsed photolysis of the photolabile chelator DM-nitrophen is important for its use in time-resolved physiological studies: the rate of substrate or effector release should be faster than the processes they initiate. Flash photolysis of DM-nitrophen using a 50-ns pulse from a frequency-doubled ruby laser (with emission at 347 nm having energy of ca. 10-20 mJ) yields short-lived photochromic or aci-nitro intermediates. At pH 6.9, double-exponential decay of a photochromic intermediate was observed for DM-nitrophen itself and its Ca2+ complex (tau 1/2 values of 24 and 570 microseconds, and 32 and 220 microseconds respectively), while only monoexponential decay of the DM-nitrophen-Mg2+ complex was detected (tau 1/2 = 31 microseconds). Only the photochemistry of DM-nitrophen-Ca2+ was found to be pH sensitive (monoexponential decay, tau 1/2 approximately 115 microseconds at pH 7.9 and 8.9). Use of the Ca(2+)-sensitive metallochromic dye antipyrylazo III in conjunction with pulsed photolysis of DM-nitrophen-Ca2+ enabled an upper limit of the half-time of release of Ca2+ to be established of ca. 180 microseconds (the rate of association of Ca2+ with the dye was probably rate determining). The rate of Ca2+ photorelease may, however, be faster than this. Thus, the DM-nitrophen-Ca2+ complex releases Ca2+ on photolysis sufficiently rapidly for the study of many Ca(2+)-dependent physiological processes with improved kinetic resolution over conventional mixing methods.

Effects of photolabile calcium chelators on fluorescent calcium indicators

The fluorescence properties of the calcium indicators Fura-2 and Fluo-3 have been investigated in the presence of the ‘caged calcium’ photolabile chelators Nitr-5 and DM-nitrophen. The excitation spectra of dilute solutions of these indicators was distorted by the presence of photolabile chelators, owing to differential absorbance of excitation light by the chelators, as well as calcium-dependent fluorescence of the chelators themselves. This distortion was altered on partial photolysis of the chelators, due to changes in their absorbance and fluorescence. At high concentrations of indicators (100 μM) and photolabile chelators (10 mM), similar to those used experimentally, DM-nitrophen quenched the fluorescence of Fluo-3 at low calcium concentrations. The results suggest that Fura-2 may be used with either chelator, and Fluo-3 with Nitr-5, to measure calcium released on photolysis of the caged compounds, but that careful calibration of the chelator-indicator mixture after the appropriate degree of photolysis is necessary.

ChemInform Abstract: A New Class of Photolabile Chelators for the Rapid Release of Divalent Cations: Generation of Caged Ca and Caged Mg.

AbstractStarting with 2‐vinyl‐4,5‐dimethoxynitrobenzene (III), prepared from the benzaldehyde (I), the chelators (VI) or (XII) are synthesized as shown in the reaction scheme.

Photolabile chelators for the rapid photorelease of divalent cations.

The properties of a recently synthesized photolabile chelator for divalent cations are described, the affinity of which for Ca2+ changes by some 5 orders of magnitude on illumination. The compound 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis[(oxycarbonyl)me thyl]-1,2-ethanediamine (DM-nitrophen) binds Ca2+ (Kd approximately 5.0 x 10(-9) M) and Mg2+ (Kd approximately 2.5 x 10(-6) M) with relatively high affinities. On exposure of the DM-nitrophen-Ca2+ complex to UV light in the 350-nm range, the chelator is cleaved yielding iminodiacetic products with a much lower affinity for Ca (Kd approximately 3 x 10(-3) M) and the free [Ca2+] increases. The quantum yield for Ca2+ release is 0.18. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with DM-nitrophen-Ca2+ complex produced maximal contraction after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in approximately 40 ms, some 5 times faster than that obtained after a rapid solution change from a Ca2+-free to a Ca2+-containing solution. In experiments with resealed human erythrocyte ghosts, irradiation of ghosts containing the DM-nitrophen-Ca2+ complex activates a Ca2+-dependent K+ efflux pathway, which is not observed in the absence of illumination. DM-nitrophen is sufficiently stable and photolabile to be used as a caged Ca (or caged Mg) for the rapid photoinitiation of divalent cation-dependent processes over a wide concentration range with a significant increase in temporal resolution over conventional mixing methods.

A new class of photolabile chelators for the rapid release of divalent cations: generation of caged calcium and caged magnesium

Synthese de derives nitro-2 dimethoxy-4,5 phenyl de l'EDTA et de l'EGTA et complexation par action de CaCl 2 ou MgCl 2