期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 2, 页码 542-547出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0710810105
关键词
hydrodynamic focusing; multiphoton microscopy; fluorescence correlation spectroscopy
资金
- NIBIB NIH HHS [9 P41 EB001976, P41 EB001976] Funding Source: Medline
- NIGMS NIH HHS [P01-GM066275, P01 GM066275] Funding Source: Medline
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 acryloclan-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 approximate to 490 mu s for the transitions in the C-terminal domain and approximate to 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.
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