title: Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer.
abstract: A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ 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 Ca2+-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 approximately 490 micros for the transitions in the C-terminal domain and approximately 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.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Hye Yoon
authors.initials: HY
authors.lastname: Park
authors.forename: Sally A
authors.initials: SA
authors.lastname: Kim
authors.forename: Jonas
authors.initials: J
authors.lastname: Korlach
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rhoades
authors.forename: Lisa W
authors.initials: LW
authors.lastname: Kwok
authors.forename: Warren R
authors.initials: WR
authors.lastname: Zipfel
authors.forename: M Neal
authors.initials: MN
authors.lastname: Waxham
authors.forename: Watt W
authors.initials: WW
authors.lastname: Webb
authors.forename: Lois
authors.initials: L
authors.lastname: Pollack
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D044085:Microfluidics; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
chemical_list: D002147:Calmodulin; D000255:Adenosine Triphosphate; D002118:Calcium