Shown). To improve cryostability and refolding glycerol was added to the

Shown). To improve cryostability and refolding glycerol was added to the solution. Additionally, to solubilize the CAB-domain, which is predicted to form a transmembrane helix, sodium cholate detergent was used, as it has a small micellar size and passes through ultrafiltration membranes. The enzyme in its in vitro refolded state, was called “FeCh refolded”. The monomeric form of His-FeCh (and also FeCh) was separated from various oligomeric forms by size exclusion chromatography (Fig. 2D); no difference in activity could be observed between pure monomer and a mixture of monomeric and oligomeric proteins. In an attempt to purify soluble His-FeCh enzyme, different E. coli expression strains (Rosetta2, BL21, C41, Origami), growth temperatures (20uC, 30uC, 37uC) and IPTG 23977191 concentrations (0.05 mM, 0.1 mM, 0.5 mM, 1 mM) were tested (data not shown). Additionally E. coli cells were stressed with 1 EtOH to force chaperone production and also a construct was created, in which maltose binding protein (MBP) was fused to the N-terminus of FeCh. Unfortunately none of these strategies were successful in yielding soluble FeCh protein (data not shown). However, coexpression in E. coli with the chaperones GroEL/GroES resulted in a considerable fraction of soluble, mature His-FeCh (1 mg/L culture) (Fig. 2B, C). This folded enzyme was called “FeCh coexpressed”. In order to compare our data with studies performed by others [32] a truncated form of FeCh (His-FeChD347), lacking the hydrophobic CAB domain, was also constructed, expressed and purified. Expression of recombinant His-FeChD347 did not result inclusion body formation and the soluble enzyme was isolated in its pure monomeric form with a molecular mass of 41.4 kDa (Fig. 2B). To be able to compare the activity of the truncated with the full-length enzyme, assays were performed on the monomeric form of FeCh and/or His-FeCh.Enzyme Kinetic Measurements Using a Continuous Fluorimetric AssayZn2+ (at a Pluripotin concentration of 0.25 to 10 mM) and freshly prepared monomeric enzyme (30 to 37 nM) were added to assay buffer (1 mL final volume) in a 161 cm cuvette and preincubated at 30uC for 5 min while stirring in a Jasco FP-6500 spectrofluorimeter with a Peltier thermostat temperature control. The assay was started by adding Proto9 and a time scan was recorded typically for 150 s with excitation at 421 nm (slit width 1 nm) and emission at 588 nm (slit width 5 nm). The initial rate was calculated from a standard curve with fluorescence at 588 nm as a SC-1 function of ZnProto9 concentration. Solutions depleted of either enzyme, Zn2+ or Proto9 were measured as controls to verify that no background signal was present. To avoid deposits of Proto9 on the cuvette walls after each measurement the cuvette was first washed with 0.05 (v/v) Tween 80 and then rinsed with MQ-water. Of the two substrates (Zn2+ and Proto9) one substrate concentration was varied, while the other concentration was kept at an enzyme saturated level. The data were plotted and fitted in Origin 6 (Microcal) to either the Hill equation (when the concentration of Zn2+ was varied) or to the Michaelis-Menten equation (when Proto9 concentration was varied). From the fitted data the kinetic parameters KM, kcat and VMAX were extracted.Pigment ReconstitutionEnzyme (either FeCh, His-FeCh, FeChD347 or His-FeChD347) was mixed with total pigment extract of Synechocystis 6803 as described previously [29] and fluorescence resonance energy transfer (FRET) was measured.Shown). To improve cryostability and refolding glycerol was added to the solution. Additionally, to solubilize the CAB-domain, which is predicted to form a transmembrane helix, sodium cholate detergent was used, as it has a small micellar size and passes through ultrafiltration membranes. The enzyme in its in vitro refolded state, was called “FeCh refolded”. The monomeric form of His-FeCh (and also FeCh) was separated from various oligomeric forms by size exclusion chromatography (Fig. 2D); no difference in activity could be observed between pure monomer and a mixture of monomeric and oligomeric proteins. In an attempt to purify soluble His-FeCh enzyme, different E. coli expression strains (Rosetta2, BL21, C41, Origami), growth temperatures (20uC, 30uC, 37uC) and IPTG 23977191 concentrations (0.05 mM, 0.1 mM, 0.5 mM, 1 mM) were tested (data not shown). Additionally E. coli cells were stressed with 1 EtOH to force chaperone production and also a construct was created, in which maltose binding protein (MBP) was fused to the N-terminus of FeCh. Unfortunately none of these strategies were successful in yielding soluble FeCh protein (data not shown). However, coexpression in E. coli with the chaperones GroEL/GroES resulted in a considerable fraction of soluble, mature His-FeCh (1 mg/L culture) (Fig. 2B, C). This folded enzyme was called “FeCh coexpressed”. In order to compare our data with studies performed by others [32] a truncated form of FeCh (His-FeChD347), lacking the hydrophobic CAB domain, was also constructed, expressed and purified. Expression of recombinant His-FeChD347 did not result inclusion body formation and the soluble enzyme was isolated in its pure monomeric form with a molecular mass of 41.4 kDa (Fig. 2B). To be able to compare the activity of the truncated with the full-length enzyme, assays were performed on the monomeric form of FeCh and/or His-FeCh.Enzyme Kinetic Measurements Using a Continuous Fluorimetric AssayZn2+ (at a concentration of 0.25 to 10 mM) and freshly prepared monomeric enzyme (30 to 37 nM) were added to assay buffer (1 mL final volume) in a 161 cm cuvette and preincubated at 30uC for 5 min while stirring in a Jasco FP-6500 spectrofluorimeter with a Peltier thermostat temperature control. The assay was started by adding Proto9 and a time scan was recorded typically for 150 s with excitation at 421 nm (slit width 1 nm) and emission at 588 nm (slit width 5 nm). The initial rate was calculated from a standard curve with fluorescence at 588 nm as a function of ZnProto9 concentration. Solutions depleted of either enzyme, Zn2+ or Proto9 were measured as controls to verify that no background signal was present. To avoid deposits of Proto9 on the cuvette walls after each measurement the cuvette was first washed with 0.05 (v/v) Tween 80 and then rinsed with MQ-water. Of the two substrates (Zn2+ and Proto9) one substrate concentration was varied, while the other concentration was kept at an enzyme saturated level. The data were plotted and fitted in Origin 6 (Microcal) to either the Hill equation (when the concentration of Zn2+ was varied) or to the Michaelis-Menten equation (when Proto9 concentration was varied). From the fitted data the kinetic parameters KM, kcat and VMAX were extracted.Pigment ReconstitutionEnzyme (either FeCh, His-FeCh, FeChD347 or His-FeChD347) was mixed with total pigment extract of Synechocystis 6803 as described previously [29] and fluorescence resonance energy transfer (FRET) was measured.