The study of cell membrane proteins has received great attention due to the important roles they play in the function of a cell. The significance of studying them also arises from the fact that membrane proteins are the common target of modern drug development. For instance, G protein-coupled receptors (GPCRs), a large family of transmembrane proteins, are involved in a wide variety of physiological processes and are the targets of over 40% of all modern medicinal drugs. Since membrane receptors usually activate signal transduction pathways through conformational changes caused by extracellular signaling events, such as ligand binding, it is crucial to understand the dynamics of these proteins in the plasma membrane. On the other hand, certain protein molecules such as RAF protein kinases usually live inside the cytosol, but get relocated to the plasma membrane during activation. As the key roles of protein kinases in cancer were identified, they have become one of the major targets for cancer drug development. For example, it was reported BRAF mutations are present in over 60% of malignant melanomas and at lower frequency in a wide range of human cancers. And recent research efforts have resulted in the development of more than a dozen of kinase inhibitor drugs including imatinib, the first kinase inhibitor drug approved for the treatment of chronic myelogenous leukemia (CML). The study of kinase activation is thus of great importance to searching compounds for effective inhibitors. In the past few decades, several single molecule techniques have been developed to study the dynamics of molecules in cell membranes. However the existing techniques either suffer from a low resolution or are not efficient for studying the dynamics of densely distributed protein molecules under physiological conditions. Here we propose to develop an efficient high-resolution nanowaveguide illuminated fluorescence spectroscopy (NIFS) technique to study the dynamics of membrane proteins. The technique is based on applying fluorescence correlation spectroscopy (FCS) to a nanoscopic excitation volume created by the near- field light exiting from the planar surface of a dielectric nanowaveguide. Unlike conventional FCS where the excitation volume is limited by the size of a diffraction-limited laser beam and typically on the order of several hundred nanometers, the proposed NIFS technique has a lateral confinement of ~40 nm and thus offers a much higher resolution. In addition, the excitation spot has a depth of ~10 nm, and is perfectly suited for studying proteins in cell membranes. Furthermore, the proposed technique is highly efficient and can characterize the dynamics of densely distributed protein molecules in cell membranes. The proposed NIFS technique thus has the potential to yield new information about the dynamics of membrane proteins as well as aid the development of innovative kinase inhibitor drugs for cancer therapy.
The research proposed in this grant application will advance a novel fluorescence spectroscopy technique to study the dynamics of cell membrane proteins with high sensitivity and efficiency. The technique can be used to study how membrane proteins respond to external stimuli at the single molecule level and thus increase our knowledge about the cellular signaling cascades. As membrane proteins are involved in many diseases including cancer, the proposed research can progress our understanding of various disease mechanisms and advance new approaches in cancer therapeutics.
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