A novel technique for determining the calcium-binding properties of the two domains of calmodulin in the presence of target peptides
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Date
2010-06
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The Ohio State University
Abstract
Calmodulin (CaM) is a ubiquitous calcium-binding protein that acts as a switch to regulate more than 500 different proteins. The classic view of CaM posits that it first binds calcium, undergoes a conformational change, and then binds to a target enzyme to alter its enzymatic activity. In order to understand the myriad of protein interactions of CaM with its targets, it is important to understand the calcium sensitivity of CaM in the presence of its targets. To measure the calcium sensitivity of these events, researchers typically utilize steady-state fluorescent techniques. However, CaM contains two different calcium binding domains located at its N-terminal and C-terminal domains. Calcium binding signals of these two domains overlap during a calcium titration, making it difficult to decipher the contribution of either domain to the binding process. To get around this, we developed a novel technique utilizing a stopped-flow apparatus that allows us to differentiate and separate the two domains in time. Stopped-flow measures the kinetics of a reaction and allows us to view the binding process as it occurs at each domain at a moment in time. By performing a sequence of stopped-flow experiments, each with a slightly different calcium concentration, we were able to simulate a steady-state titration, yet beneficially view each domain as a separate, non-overlapping entity. Thus, we can now successfully observe the calcium sensitivity of each CaM domain in the presence of a target, something that was previously very difficult to do. This technique will allow further research into CaM binding, leading to greater insight into the workings of this protein. Advisor: Dr. Jonathan P. Davis
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Keywords
calmodulin, stopped-flow, myosin light chain kinase, calcineurin, cam kinase II