Key Points
- To synthesize and characterize a panel of coumarin-labeled ATP and ADP analogues with varying linker lengths to optimize fluorescence signals when probing P-loop ATPase mechanisms.
- Synthesized ATP and ADP analogues with three coumarin fluorophores conjugated to the 3'-position of the ribose via propylenediamine, ethylenediamine, or direct zero-length linkers using combined chemical and enzymatic routes.
- Assessed fluorescence properties and binding/dissociation kinetics in interactions with rabbit skeletal muscle myosin subfragment 1 in both the presence and absence of actin.
- The zero-length linker analogue deac-aminoATP generated a 17-fold fluorescence enhancement upon binding to skeletal myosin II.
- The diphosphate form of deac-aminoATP produced a large signal change during dissociation from actomyosin while maintaining kinetic behavior comparable to natural ADP.
Structured PICO
PPopulationRabbit skeletal myosin subfragment 1 in the presence and absence of actin
IInterventionSynthesized ATP and ADP analogues with fluorophores linked via the 3'-position of the ribose moiety (e.g., deac-aminoATP)
OOutcomeFluorescence properties and kinetics of interaction with myosin
The synthesized fluorescent nucleotide analogues produce large signals upon interaction with actomyosin, indicating potential utility for studying scarce proteins in small amounts.