The goal of the present project is to obtain high-resolution structural information on ionotropic glutamate receptors (GluRs), multi-subunit ligand-gated cation channels which mediate the fast synaptic excitation in the brain. Resolving the structure of the GluRs should provide the basis for understanding excitatory synaptic signalling and tools to develop new therapeutic approaches. By content and objectives this project complies with the objectives described Research Area 4.5.
The specific objectives are:
1. to prepare purified GluRs and their functional ligand binding domains in a state suitable for detailed structural studies
2. to determine the three-dimensional structures of the soluble ligand-binding domain (X-ray crystallography) and of the intact glutamate receptors (single-particle electron microscopy and electron crystallography of 2D crystals)
3. to study structure-function relations in GluRs and GluR fragments by a variety of biophysical and biochemical tools, including panels of binding reagents generated by phage display technology
This project invokes tightly coupled research activities from four European laboratories: 1. Dr K. Keinanen (VTT), Espoo, FI; 2. Dr B. Sakmann (MPIMF), Heidelberg, DE; 3. Dr J. Rossier (CNRS 2054), Paris, FR; 4. J.Engberg (RDSP), Copenhagen, DK. These laboratories collaborate already actively, and the current status of the research provides an excellent platform to address the structure of glutamate receptors. In particular, we have recently demonstrated that a functional ligand binding domain of an AMPA-selective glutamate receptor can be produced by recombinant expression as a water-soluble, secreted protein. This separation of the binding site from the membrane-embedded domains is unique among neurotransmitter receptors and provides an opportunity to study the structural basis of ligand binding by X-ray crystallography. Also, we have developed protocols for high-level expression, solubilization and purification of intact recombinant GluRs.
The availability of milligram amounts of both soluble ligand-binding domains and intact integral membrane channels allows us for the first time to directly study the three-dimensional structure of GluRs.
In pursuit of this structural information, single particles of intact, purified GluRs will be analysed by electron microscopy to determine receptor subunit stoichiometry and the overall shape of the receptor.
Attempts will be made to obtain 2- and 3-dimensional crystals of both the soluble domain and the intact receptor for electron or X-ray crystallography, as a basis for structural studies at atomic resolution. At the same time, we will perform a detailed biochemical and biophysical analysis of both the proteins itself.
Specific binding reagents generated by the phage display technology will be used as central tools to study the functional and structural topology of GluRs to produce complementary structural information. As minimization of protein heterogeneity is critical for successful crystallization, our project will also include a systematic protein chemical study to assess the biochemical homogeneity of the isolated material, which helps us to further develop the expression, solubilization and purification protocols.
Funding SchemeCSC - Cost-sharing contracts
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