The discovery of the green fluorescent protein (GFP), and its applications have revolutionized many fields of life sciences. The GFP (and other fluorescent proteins, XFPs) have now been used as a basis for development of optical sensors of a wide range of cellular properties and processes. In order to convert changes in cellular properties into an optically detectable signal, the currently used probes exploit only one mechanism: environment-induced changes of the optical properties of the fluorophore Our theoretical work suggests that another class of probes exists, and provides the theoretical background for development and use of these probes. The new class of probes utilizes the fact that the XFP fluorophore is planar, and thus its optical properties are anisotropic. If the anisotropic XFP fluorophore is anchored to non-centrosymmetric support, such as to the cell membrane, under the right conditions even small changes in fluorophore orientation should lead to pronounced changes in observed fluorescence. Our experimental work on development of a voltage-sensitive fluorescent protein (VSFP) provides preliminary evidence for existence and usability of this phenomenon. It is the goal of the proposed work to test the hypothesis that fluorophore anisotropy can serve as a basis of a range of genetically encoded sensors of membrane protein activity and structure. To test this hypothesis we aim to (1) prove that the observed fluorescence changes in our VSFP constructs are due to changes in fluorophore orientation; (2) deduce information about the structure of our VSFP construct from our fluorescence imaging data; and (3) apply our methods to other proteins. The new mechanism of transduction of conformational changes to changes in fluorescence should lead to an improved VSFP, and to development of a number of genetically encoded reporters of activity and structure of membrane proteins, with a large scientific impact.
Field of science
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins
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