Park Hye Yoon, Kim Sally A, Korlach Jonas, Rhoades Elizabeth, Kwok Lisa W, Zipfel Warren R, Waxham M Neal, Webb Watt W, Pollack Lois
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):542-7. doi: 10.1073/pnas.0710810105. Epub 2008 Jan 4.
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.
一种微流体混合器被用于研究钙调蛋白在结合Ca2+时构象变化的动力学。该装置通过将流体动力学聚焦与扩散混合解耦，实现了快速、均匀的混合，并能达到数十微秒的时间尺度。该混合器与多光子显微镜结合使用，以检测丙烯罗丹明标记的钙调蛋白由Ca2+诱导的快速转变。我们发现，两个同源球状结构域中构象变化的动力学速率相差一个多数量级。蛋白质C端结构域转变的特征时间常数约为490微秒，而N端结构域转变的特征时间常数约为20毫秒。我们讨论了这两个不同事件的可能机制以及稳定中间体——半饱和钙调蛋白的生物学作用。
