Cell signaling is at the core of most biological functions and often involves dynamic interactions among proteins. Protein-protein interactions induce conformational changes that initiate chain reactions, which in turn lead to cellular responses. A comprehensive understanding of such protein interactions is crucial to understanding the regulatory mechanisms controlling cellular functions. To study protein interactions in cell signaling, ensemble measurements, which yield information only on averaged properties, are inadequate. Intrinsically, the crucial early events of cell signaling often involve only a few molecules and then are magnified along the signaling pathways. Moreover, for intrinsically heterogeneous systems such as protein complexes, protein interaction dynamics contain static and dynamic disorders as a result of spatial and temporal inhomogeneities. Stochastic protein-protein interactions prevent such characterizations when many molecules are measured simultaneously. Single-molecule spectroscopy is crucial to obtaining such information because it is capable of characterizing biomolecular processes that are inhomogeneous and non-synchronizable. We have conducted a single-molecule study of protein-protein interaction dynamics in an intracellular signaling protein complex, Cdc42/WASP, employing a novel CBD biosensor, which is a dye-labeled WASP fragment that binds only the GTP-activated Cdc42, we were able to probe hydrophobic interactions associated with Cdc42/WASP recognition. Single-molecule fluorescence measurements indicated static and dynamic disorders in this protein-protein interaction system, and our study characterized the dynamic and inhomogeneous nature of molecular recognition within the Cdc42/WASP signaling complex. A coupled-two channel fluctuation Markovian model was proposed for the fluctuation dynamics, and the molecular structure and solvent-accessible surface of the protein-protein interaction complex were explored by MD simulations. The Cdc42-CBD complexes showed conformational fluctuations between bound and loosely bound states while the overall complex was still associated. The distribution of the fluctuation rates was highly inhomogeneous, with variations among individual complexes of two orders of magnitude under the same conditions. The results suggest highly dynamic rather than static protein-protein interactions in this cell signaling system. Such dynamic and fluctuating protein-protein interactions may be important for inducing and regulating downstream signaling events. By establishing a molecular imaging system with adequate spatial and temporal resolution and combining single-molecule experimental and computational approaches, we expect to expand our studies to other important biomolecular complexes under physiological conditions and eventually to living cells.
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