Calmodulin Mediated Disorder-to-order Transitions: Calcineurin as a Model System

University Of Kentucky Research Foundation, Lexington KY

Investigators

Abstract

In recent years many intrinsically disordered proteins have been identified in numerous biologically important systems. An intrinsically disordered protein is one that is entirely or partially unstructured. It is not clear is why protein disorder is so common. A number of hypotheses have been advanced. The hypothesis being tested in this project is that disorder-to-order transitions can be used to regulate protein function. Calmodulin is a very abundant, highly conserved calcium-binding protein known to regulate the functions of numerous essential other proteins. It was recently proposed that many of the calmodulin binding sites within target proteins are located in intrinsically disordered regions and that some of these undergo disorder-to-order transitions upon calmodulin binding. Calcineurin is an essential, highly-conserved serine/threonine phosphatase that is activated when bound by calmodulin. Upon binding of calmodulin to the regulatory domain of calcineurin, a conformational change is induced that results in activation of the enzyme. Experimental data suggests the regulatory domain of calcineurin is disordered in the inactive enzyme, and that it undergoes a disorder-to-order transition upon calmodulin binding. The goal of this research is to characterize the physical nature of this disorder-to-order transition. Calcineurin is being employed as a model system in studies of calmodulin-mediated disorder-to-order transitions that regulate biological activity. This goal will be achieved through two aims: i) determination of the ability of the regulatory domain to undergo a calmodulin mediated disorder-to-order transition as an isolated domain, and ii) determination of the role full length calcineurin plays in this transition. A variety of biophysical techniques will be employed, including circular dichroism, fluorescence, NMR, and analytical ultracentrifugation. The data obtained will enable construction of a model for the calcineurin regulatory domain disorder-to-order transition, which in turn will be used as the basis for future studies of other calmodulin mediated transitions involved in protein regulation. The central question in the field of intrinsically disordered proteins is why is disorder so common? This project will make a significant contribution towards answering part of that question through the testing of the hypothesis that calmodulin mediated disorder-to-order transitions can regulate protein function. The PI plans to have both a postdoctoral scholar and several undergraduate students work on aspects of this project. Where possible, these will include representatives from underrepresented groups. The postdoctoral scholar will receive advanced training in protein chemistry, molecular biology, spectroscopy and NMR spectrometry. The undergraduate students will receive basic laboratory training, training in protein chemistry, molecular biology, spectroscopy, scientific ethics, and in the analysis and presentation of data. All trainees will participate in the dissemination of knowledge generated. The PI will incorporate knowledge generated in this project into graduate-level courses on structural biology and proteins. In addition, data will be published in peer-reviewed journals, and presented at scientific meetings and research institutions.