The long-term goal of this proposal is to understand the mechanism of how calmodulin (CaM) selects among hundreds of its potential binding targets (CaMBTs) in vitro, so that novel strategies can be developed to direct the selection of specific binding partners in vivo. Given CaM's role in the regulation of the cell cycle and apoptosis, such strategies may well lead to the ability to regulate signaling pathways that allow suppression of uncontrolled cellular growth or to induce apoptosis. The objective of the present proposal is to characterize different structural states of CaM and to determine how intracellular factors (e.g., Ca2+ binding and macromolecular crowding) influence its target-binding and recognition using a combined computational, experimental and bioinformatics approach. Our hypothesis is that there is a sequence-structure-function- environment relationship in CaM that influences the specific types of targets with which it interacts. The rationale for the proposed research is that once we characterize the free energy landscape of binding and recognition and their kinetics for CaM in response to various factors in a cell-like environment, we can design strategies that are able to control the formation of selected CaMBTs as well as to expand a web-based database of CaM/CaMBT complexes additionally annotated with this new data and by cellular localization. We will test our central hypothesis by pursuing the following three specific aims: (1) Characterize the free energy landscape of target binding and recognition of CaM by computer simulations in solvent conditions relevant to those found inside cells. (2) Experimentally determine the conformational states populated by CaM in solvent conditions relevant to those found inside cells and examine how the altered spatiotemporal distribution of states influences Ca2+- and target-binding. (3) Annotate the structural conformation of CaM/CaMBT complexes in different cellular conditions with bioinformatics approaches. The research proposed is innovative because it combines the approaches of multiscale molecular dynamics simulation, spectroscopy, and bioinformatics in the characterization of CaM structures for binding and recognition and the annotation of CaM's biological relevance in a cell. The contribution is significant because it is the first step in a continuum of research that is expected to reveal new strategies regarding the selection and manipulation of CaM binding where one could potentially guide choices between, for example, cell division and apoptosis. The proposed research is of significance because the knowledge gained regarding the control of the properties of CaM in a cell is expected to expand the fundamental understanding of other signaling proteins that depend on conformational flexibility for their function. Such mechanistic insights could well lead to innovative approaches in cell cycle control and perhaps therapeutic strategies for cancer treatment.
The proposed research is relevant to public health because when the strategies to manipulate signaling proteins such as calmodulin become available, there is the promise that cell growth and cell death can be controlled by appropriately perturbing the cellular environment to direct the activation or suppression of the appropriate calmodulin signaling pathways. This is relevant to NIH's mission because important advances in novel therapeutic strategies associated with uncontrolled growth and spread of abnormal cells could be expected.
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