MEMSENSORS

A novel class of genetically encoded sensors of membrane protein function and structure

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 Obiettivo del progetto (Objective)

'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.'

Introduzione (Teaser)

The green fluorescent protein (GFP) exhibits bright green fluorescence when exposed to blue light. It is frequently used as a reporter of expression; in modified forms it can also be used to develop biosensors.

Descrizione progetto (Article)

Discovery of the green fluorescent protein (GFP) has led to many applications in cell and molecular biology, revolutionising many areas of life sciences. The GFP and other fluorescent proteins (XFPs) are useful in the development of optical sensors, which provide information on a wide range of cellular properties and processes. However, probes currently in use only consider environmental changes of the optical properties of fluorophores in order to convert changes in cellular properties into an optically detectable signal.

'A novel class of genetically encoded sensors of membrane protein function and structure' (Memsensors) set out to prove that another class of probes exists by providing the theoretical background for development and use of such probes.

The new class of probes exploits the planar properties of the XFP fluorophore since its optical properties are anisotropic, i.e. dependent on direction. When anchored to a cell membrane, for instance, even small changes in fluorophore orientation should offer marked changes in observed fluorescence. Project partners set various objectives to prove that fluorophore anisotropy can serve as the basis for a variety of genetically encoded sensors of membrane protein activity and structure. The development of a voltage-sensitive fluorescent protein (VSFP) gave preliminary evidence for the existence and applicability of this phenomenon.

The EU-funded project's accomplishments show that fluorophore anisotropy can be observed in a substantial number of membrane proteins in living cells. Researchers created an imaging technique for conducting experiments that produced quantitative information about membrane protein structure and function. Applicable to a large number of proteins, this achievement has major scientific and commercial significance. The technique can facilitate the development of a usable VSFP to detect voltage pulses carrying information in living neurons. This outcome has the potential to revolutionise activities in the field of neuroscience research.

Another important objective was realised with the creation of a device that enables precise monitoring of conformational changes in proteins on a sub-millisecond timescale. This device and its microscopic method have been awarded a Czech patent, and its application is under consideration for a Patent Cooperation Treaty (PCT) patent.

project results were presented at various scientific gatherings, and a number of scientific publications have been submitted. Memsensors project outcomes have the potential to revolutionise activities in the field of neuroscience research.