Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10533761 | Analytical Biochemistry | 2005 | 8 Pages |
Abstract
We have developed assays for the binding of nucleotide and protein substrates to p38α protein kinase based on time-resolved Forster resonance energy transfer. p38α was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin “LANCE” europium chelate conjugate and p38α. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100 pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent Kd decreases with a relaxation time of 21 min at 125 pM biotin-p38α. Preincubating inhibitors or ATP with biotin-p38α and adding M39AF as a competitor yielded IC50s consistent with those measured by enzyme assay for the activated form of biotin-p38α. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38α. To measure affinity of p38α for its protein substrate MK2, we incubated biotin-p38α with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38α and detection of 0.5 nM p38α · MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38α yielded an IC50 value of 5 nM. Activation of either p38α or MK2 had no effect on the measured Kd. M39AF was found to bind in a ternary complex with p38α · MK2 with lower affinity than that observed in the binary complex with p38α alone.
Related Topics
Physical Sciences and Engineering
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Analytical Chemistry
Authors
Wen Xiao Zhang, Ruixiu Wang, Douglas Wisniewski, Alice I. Marcy, Philip LoGrasso, Jean-Marie Lisnock, Richard T. Cummings, James E. Thompson,