The overall goal of this project is to detect single calmodulin(CaM) molecules as they function. Key questions need to be answered about how CaM recognizes and binds to a target. CaM is a key link in many biochemical calcium signaling pathways. The flexible structure of CaM and the wide variety of potential targets suggests that a distribution of conformations exists at the molecular level. Single-molecule experiments are therefore proposed in order to investigate conformational fluctuations and heterogeneity of CaM and CaM-target complexes. Whereas conventional methods measure an ensemble average, single-molecule measurements probe individual variations in structure and target activation. Preliminary investigations are described that have uncovered a distribution of binding conformations for CaM bound to the CaM-binding domain of the plasma membrane Ca-ATPase (PMCA). It is suggested that this distribution correlates with a distribution of activity levels of the enzyme, providing a mechanism for fine-tuning of enzyme regulation. This proposal seeks to test this hypothesis. The proposal also seeks to understand the molecular mechanisms of target recognition and binding by detecting individual binding events. Such measurements are needed to probe targets. The proposed research will also measure the coupling between CaM-binding and enzyme activation in the PMCA, permitting a detailed investigation of this mechanism. An important step in the project will be developing methods to immobilize the protein during observation and implementing fluorescence probes of single-molecule binding and activation. Because calmodulin regulates numerous biological processes, this work is pertinent to a wide range of health concerns, including neurotransmission and learning, oxidative damage and aging, and muscle activation. An understanding of the interaction between calmodulin and target proteins under conditions involving cellular signaling or oxidative stress will be crucial in designing therapies to alleviate disorders involving these processes. The experimental approach will combine recently developed techniques of single-molecule spectroscopy with fluorescence resonance energy transfer and site-directed mutagenesis as a probe of protein structure and dynamics.
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