Manipulating Signaling Proteins for Target Binding and Recognition

描述（由申请人提供）：本提案的长期目标是了解钙调素（CaM）如何在体外从数百个潜在结合靶点（CaMBTs）中选择的机制，以便开发新的策略来指导体内特异性结合伴侣的选择。鉴于钙调素在调节细胞周期和细胞凋亡中的作用，这种策略可能会导致调节信号通路的能力，从而抑制不受控制的细胞生长或诱导细胞凋亡。本提案的目的是表征CaM的不同结构状态并确定细胞内因子（例如，Ca 2+结合和大分子拥挤）影响其靶点结合和识别，使用结合计算，实验和生物信息学的方法。我们的假设是，有一个序列-结构-功能-环境的关系，在钙调素的影响与它相互作用的特定类型的目标。所提出的研究的基本原理是，一旦我们表征了结合和识别的自由能景观及其动力学的钙调素在细胞样环境中响应于各种因素，我们可以设计的策略，能够控制选定的钙调素的形成，以及扩大基于网络的数据库的钙调素/钙调素复合物，另外注释了这个新的数据和细胞定位。我们将通过追求以下三个具体目标来测试我们的中心假设：（1）通过在与细胞内发现的溶剂条件相关的溶剂条件下的计算机模拟来表征CaM的靶结合和识别的自由能景观。(2)实验确定的构象状态由钙调素填充在溶剂条件下相关的细胞内发现的，并检查如何改变时空分布的状态影响Ca 2+和目标结合。(3)用生物信息学方法诠释不同细胞条件下CaM/CaMBT复合物的结构构象。提出的研究是创新的，因为它结合了多尺度分子动力学模拟，光谱学和生物信息学的方法，在表征钙调素结构的结合和识别和钙调素的生物相关性在细胞中的注释。这一贡献是重要的，因为它是连续研究的第一步，预计将揭示关于选择和操纵钙调素结合的新策略，其中一个可能指导选择，例如，细胞分裂和凋亡。拟议的研究是有意义的，因为所获得的知识有关的控制在细胞中的钙调素的性质，预计将扩大其他信号蛋白，依赖于构象的灵活性，为他们的功能的基本理解。这种机制的见解很可能导致细胞周期控制的创新方法，也许是癌症治疗的治疗策略。

DESCRIPTION (provided by applicant): 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.