Conformational changes of calmodulin upon Ca 2+ binding studied with a microfluidic mixer

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Abstract
A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca 2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca 2+ -induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are ≈490 μs for the transitions in the C-terminal domain and ≈20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.