Our goal is to understand the molecular mechanisms of synaptic plasticity.

Ca2+ signaling is the hub of these biochemical mechanisms. We are currently assessing the decoding of Ca2+ signals into integrated biological responses through the third messenger molecule calmodulin. Calmodulin binds four Ca2+ ions.  Through conformational changes, Ca/calmodulin binds to and activates a variety of effector molecules, including enzymes such as protein kinases and phosphatases. One of our major focuses is the kinetics of calmodulin binding to the family of calmodulin-dependent enzymes.  Fluorescent reagents permit us to measure the association and dissociation kinetics of calmodulin with target enzymes using steady-state and stopped-flow fluorescence techniques.

Steady-state measurements are accomplished on a T-based PTI Quantamaster fluorimeter running Felix 32 software. The T-based format streamlines fluorescence polarization and anisotropy measurements.

Stopped-flow measurements are made on an Applied Photophysics Limited SX.18MV sequential stopped-flow fluorimeter capable of measuring association and dissociation rates on the millisecond timescale (right). This instrument resides in the laboratory of Dr. John Putkey, a long time collaborator on these projects.

Figure 1.  Click to enlarge                     

Calmodulin Interactions with CaM Kinase II

CaM Kinase II (CaMKII) is activated by Ca2+ /calmodulin and can phosphorylate itself when activated . To quantify these interactions, calmodulin was labeled with acrylodan, a probe that exhibits increased florescence when calmodulin binds to Ca2+ and when Ca2+ binds to target proteins. This environmentally sensitive probe is one member of a family of molecules that we have used to examine interactions between calmodulin and target proteins.

Using steady-state and stopped-flow fluorescence we have quantified the kinetics of these interactions. Data from one such experiment is shown in Figures 2 and 3. Dissociation of the fluorescently labeled calmodulin from CaMKII in the presence of Ca2+ shows a dissociation rate of 1.6 s -1 (Figure 2). When CaMKII is autophosphorylated, the dissociation rate of calmodulin decreases dramatically to 2 x 10 -5 s -1 (Figure 3).

Figures 2 (top) and 3.  Click on images

to enlarge.