Objective
We propose to develop an integrated microscopical approach to probe structure-function relationships of large uncrystallised macromolecular assemblies of high resolution. We aim at elucidating both the structural and the functional aspects of the molecules at the resolution level of 12 nm. Our problem-oriented methodology development is directly supported by the availability of recombinant protein (in its wild type and in mutated forms). Our test system is the type 3 isoforrn of the ryanodine receptor calcium release channel family. The integrated approach synergetically combines: 1) Three-dimensional (3D) cryo-electron microscopy of single macromolecules embedded in vitreous ice using the "angular reconstitution" method [Serysheva et al., 1995]. One objective is to push the attainable resolution to the 1 nm level, which resolution is feasible [R. Henderson, Quart. Rev. Bio phys 28 (1995) 171-193]. A Simple Super Computer (SSC) consisting of an array of Pentium-based computers (to be programmed by subcontractor Image Science Software GmbH) will provide the necessary computing resources. 2) Molecular Recognition Force Microscopy (MRFM, Hinterdorfer et al., 1996). Atomic force microscopy (AFM) [Z. Shao, J. Yang, Quart. Rev. Biophys 28 (1995) 195-251] is combined with molecular recognition by attaching an antibody to the AFM tip via a flexible linker molecule. This novel type of scanning probe microscope will be used to map antigenic sites in the RyR3 protein. 3) Single Dye Tracing (SDT, Schmidt et al., 1995) for time-resolved (ms range) imaging of single fluorescent molecules (label on antibodies against RyR3 in this proposal) within a membrane. This technique can be combined with the patch-clamp techniques which combination allows the measurement of the functional activities of single channels while quantitatively observing the behavior of the dyes (angular and lateral motion, co-localization). The combination of the above techniques (Partners 1 and 3), in close interaction with the supporting biochemistry (Partner 2) providing specific clones and antibodies, will allow unprecedented insights into structure-function relations at resolution levels to 1 nm. Although X-ray crystallography may provide resolutions of 0.3 nm (assuming one manages to crystallise the protein), our approach (which uses no crystals at all) provides fast structural determinations of different isoforms in various conformations and allows direct correlations between functional activity and 3D structural organization.
The partners in this project are international leaders who have made major discoveries in their fields.
The Linz group has pioneered the MRFM and the SDT technologies. The London group has invented the angular reconstitution approach with which a number of 3D structures were recently determined. The Milano group was the first to describe the RyR3 isoform.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences computer and information sciences software
- natural sciences earth and related environmental sciences geology mineralogy crystallography
- natural sciences chemical sciences inorganic chemistry alkaline earth metals
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences physical sciences optics microscopy
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Coordinator
4040 Linz - Auhof
Austria
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