Detection in Living Cells of Ca2+-dependent Changes in the Fluorescence Emission of an Indicator Composed of Two Green Fluorescent Protein Variants Linked by a Calmodulin-binding Sequence

Abstract

MATERIALS AND METHODS Expression and Purification of Proteins-The vector for expression of FIP-CB SM is similar to the one described by Mitra et al. (5). The coding sequences for the BGFP (6) and RGFP (7) domains were produced by amplifying the GFP-encoding sequences in the BioBlue™ and BioYellow™ vectors obtained from PharMingen, Inc. (San Diego, CA). The vector pETIC, encoding a fluorescent indicator protein (FIP) control consisting of RGFP and BGFP domains joined by the linker sequence, GTSSGSSTGA, was generated first. The RGFP domain in pETIC is fused to the His 6 -tag/thrombin/S-tag/enterokinase leader sequence derived from pET30a (Novagen, Inc., Madison, WI). The C terminus of the BGFP domain is fused to an additional His 6 sequence, also derived from pET30a. The vector pETIC-1 encodes FIP-CB SM , which is identical to the FIP control, except that the linker has the sequence: GTSSRRKWNKTGHAVRAIGRLSSTGA. Boldface type denotes the CaM-binding sequence from avian smooth muscle myosin light chain kinase (8).For expression, pETIC and pETIC-1 were transformed into Escherichia coli strain BL21(DE3). Cells containing pETIC or pETIC-1 were grown at 23°C to an A 600 of 0.6 -0.8, and protein expression was induced by addition of isopropyl-1-thio-␤-D-galactopyranoside to 0.5 mM. After incubating at 23°C for ϳ40 h, cells were harvested. Control FIP and FIP-CB SM were purified using His 6 affinity chromatography essentially as described by Mitra et al. (5). Vertebrate CaM expressed in E. coli was purified as described previously (9). The concentrations of control FIP and FIP-CB SM were determined using an ⑀ 490 of 89 mM Ϫ1 cm Ϫ1 . Concentrations of FIP-CB SM stock solutions were verified by titration with a standard CaM solution.In Vitro Measurements of FIP-CB SM Fluorescence-Fluorescence measurements were performed using a Photon Technology International (Monmouth Junction, NJ) QuantaMaster™ photon counting spectrofluorometer. Reaction volumes (3 ml) were incubated at 30°C in a stirred cuvette. Excitation and emission slit widths were 5 nm. An excitation wavelength of 380 nm was used for in vitro measurements of FIP fluorescence. For most experiments, a buffer containing 25 mM Tris, 0.1 M NaCl, and 300 M CaCl 2 , pH 7.5, was used. For experiments in which the free Ca 2ϩ ion concentration was varied, a buffer containing 50 mM Tris, 0.1 M NaCl, 0.5 mM MgCl 2 , and 3 mM 1,2-bis(2-amino-5,5Ј-dibromophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, pH 7.5, was used. Aliquots of standard CaCl 2 solutions were added to achieve various levels of free Ca 2ϩ ion, which were calculated using the MaxChelator program (10). The FIP-CB SM fluorescence emission spectrum is essentially independent of the pH between 7.0 and 8.0, either in the presence or absence of bound (Ca 2ϩ ) 4 -CaM.

Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1

“…Alternatively, it is possible that there is, in essence, no pool of "free" CAM to compete with the newly synthesized protein. At elevated [Ca 2ϩ ] i , this condition appears to apply, with free (non-target bound) CAM representing about 0.1% of total CAM protein (40). The situation at resting [Ca 2ϩ ] i is less well understood.…”

Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

“…This approach has been widely adopted to measure the activity of proteases that are activated during apoptosis (Rehm et al 2002;Takemoto et al 2003). More sophisticated approaches are usually necessary to develop sensors for kinase activity, nucleotide exchange activity or small molecules, such as calcium (Miyawaki et al 1997;Romoser et al 1997), cAMP (Ponsioen et al 2004) and DAG (Violin et al 2003;Sato et al 2006).…”

Calcium and signal transduction

“…Another group of Ca 2þ indicators, which is still in the initial stages of development, operates on fluorescence resonance energy transfer (FRET) technology. Cameleons and pericams [34,35] are proteins that consist either of two differently colored mutant green fluorescent protein (GFP) molecules, or of one molecule divided in two parts and separated by a molecule of CaM which serves as molecular switch. As mentioned earlier, CaM changes its conformation when binding Ca 2þ , bringing the GFP molecules within FRET distance.…”