Anti-His tag antibody [DyLight® 488]

ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA(+)-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA(+)-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3 ')Y26C, is reported. The protein is part of the alpha Rep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific alpha Rep bA3-2 that is known to induce the opening of the (A3A3 ') interdomain region and c) a derivative of the alpha Rep bA3-2 inducer extended with a His(6)-Tag (His(6)-bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His(6)-bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein-ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.