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<br>The substrates were <i>p­</i>-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.<br>The effectors were the competitive inhibitors phosphate, molybdate, tungstate, arsenate, and vanadate, as well as magnesium ion. These were used with the substrate amifostine.<br>The 12-nucleotide nanoantenna used here has biotin at 5′ and fluorescein (FAM) at 3′. The excitation (λ_ex) and emission (λ_em) 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.<br>See associated paper for details about the procedure and reagents.