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.

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A primary mechanism for initiating smooth muscle contraction involves an increase in [Ca 2ϩ ] i , leading to myosin regulatory light chain (RLC) phosphorylation, crossbridge cycling, and force development (1, 2). Phosphorylation of Ser-19 on RLC of myosin II changes the orientation of myosin crossbridges, allowing actin activation of myosin ATPase activity. A similar mechanism occurs with nonmuscle myosin II with effects on many cellular actomyosin-dependent functions. The Ca 2ϩ -dependent phosphorylation of RLC is mediated by Ca 2ϩ ͞cal-modulin (CaM)-dependent myosin light chain kinase (MLCK), whereas myosin light chain phosphatase dephosphorylates RLC. In smooth muscles, agonists stimulate greater RLC phosphorylation and force than do depolarizing stimuli at comparable [Ca 2ϩ ] i because of Ca We developed a different CaM-sensor MLCK capable of monitoring MLCK activation to obtain temporal and quantitative information on Ca 2ϩ ͞CaM binding to MLCK where Ca 2ϩ -dependent CaM binding increased kinase activity coincident with a decrease in FRET (11). The CaM-sensor MLCK was expressed in smooth muscle tissues of transgenic mice to obtain quantitative insights on CaM activation of MLCK relative to [Ca 2ϩ ] i and RLC phosphorylation and force development. These results show that genetically encoded biosensors may be used to investigate physiological processes in tissues of transgenic mice. MethodsConstruction of SM8 35 KCS Plasmid. The pSM8 35 KCS construct was prepared by subcloning the 1.6-kb cDNA of Ca 2ϩ ͞CaM-sensor containing the MLCK CaM-binding sequence flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (12) into the pSM8-CAT vector, which contains the smooth muscle ␣-actin promoter (13, 14). The 3.1-kb cDNA fragment of short rabbit smooth muscle MLCK was further subcloned into the site between the Ca 2ϩ ͞ CaM-sensor gene and the smooth muscle ␣-actin promoter in pSM8-CAT vector to produce pSM8 35 KCS construct. Correct This paper was submitted directly (Track II) to the PNAS office.

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