Monitoring protein conformational changes using fluorescent nanoantennas

Fluorescent nanoantennas generated a transient fluorescence "spike" during the hydrolysis of a substrate by calf intestinal alkaline phosphatase (AP). With AP, these fluorescent nanoantennas were used to characterize:1) the hydrolysis of 15 substrates2) the hydrolysis of one substrate in the presence of six effectors
The substrates were p­-nitrophenylphosphate (pNPP), 4-methylumbelliferylphosphate (4MUP), pyrophosphate (PPi), β-glycerophosphate (BGP), phosphoenolpyruvate (PEP), L-phosphoserine (PSer), pyridoxal 5'-phosphate (PLP), D-glucose-6-phosphate (G6P), D-fructose-6-phosphate (F6P), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate (GTP), phosphocreatine (PCr), and amifostine.
The effectors were the competitive inhibitors phosphate, molybdate, tungstate, arsenate, and vanadate, as well as magnesium ion. These were used with the substrate amifostine.
The 12-nucleotide nanoantenna used here has biotin at 5′ and fluorescein (FAM) at 3′. The excitation (λex) and emission (λem) wavelengths were 498 nm and 520 nm, respectively. Conditions were [nanoantenna] = 150 nM, [streptavidin] = 50 nM, [biotinylated AP] = 20 nM, [substrate] = 300 μM, and as applicable, [inhibitor] = 30 μM or [Mg2+] = 5 mM. The buffer was 100 mM Tris, 10 mM NaCl, pH = 8.0, and temperature = 37 °C. All measurements were recorded in triplicate.
See associated paper for details about the procedure and reagents.