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Probing conformational dynamics in signalling EGF/ErbB receptors via single-molecule multidimensional fluorescence microscopy and molecular modelling

Reference	BB/C51464X/1
Principal Investigator / Supervisor	Professor Marisa Martin-Fernandez
Co-Investigators / Co-Supervisors	Dr Barry Barraclough, Dr David Clarke, Dr Anthony Fordham-Skelton, Dr ian gould, Dr M Tobin, Dr Stephen Webb
Institution	STFC - Laboratories
Department	Central Laser Facility (CLF)
Funding type	Research
Value (£)	256,413
Status	Completed
Type	Research Grant
Start date	16/05/2005
End date	15/05/2008
Duration	36 months

Abstract

The proposed programme of work aims to elucidate the mechanism by which growth signals are transduced across the plasma membrane by ErbB receptors and how this leads to the stimulation of the receptors catalytic activity that ultimately results in cell division. Growth factor receptor signalling is a single molecular event mediated by its interaction with other receptors and or by intra-receptor structural reorganisation. High-resolution observations are therefore required to understand receptor-mediated signal transduction. The techniques currently available do not allow determination of crucial aspects of receptor signalling, such as the number and nature of interacting receptor partners and or the conformational changes in receptor complexes during signal transduction in the cell. These observations are masked by ensemble and time averaging and by poor spatial resolution. The required resolution can only be provided by X-ray crystallography, but this provides only brief structural snapshots from which receptor dynamics can not be inferred. Achieving our objectives therefore require: 1. The optimisation of imaging technologies we have recently developed based on the analysis of time-resolved fluorescence data from single-molecules in the plasma membrane of living cells. This technique employs total internal reflection illumination and collects multidimensional single-molecule fluorescence imaging data in video rate time frames to simultaneously measure the time courses of single pair fluorescence resonance energy transfer (spFRET) and single molecule fluorescence polarisation (smFP) changes. The combination of spFRET and smFP data from individual fluorophores can provide quantitative information on inter- and intra-receptor distances, and on the axial and azimuthal angles of extracellular and intracellular receptor domains during signalling in the living cell. The single-molecule technique can therefore be used to determine the stoichiometry and conformational dynamics of growth factor ErbB receptor complexes during signalling. These data cannot be simultaneously obtained by any other method. Unlike ensemble-averaged measurements, spFRET and smFP can be measured simultaneously from each receptor on a physiologically relevant population in the cell, and can therefore potentially reveal transient ErbB conformation intermediates, even those with steady state concentrations too low to measure using ensemble methods. Determining heterogeneity and degree of fluctuations is crucial to understand ErbB function because seemingly identical copies of a protein do not necessarily have the same function in the cell [ref]. 2. Combining the experimental data on receptor conformational changes with molecular dynamics modelling to probe interactions in ErbB homo- and heterodimers with respect to their conformational stabilities. Marrying quantitative results on receptor distances and domain orientations with modelling at atomic resolution can potentially lead to the full elucidation of the mechanisms of signalling of both wild-type and oncogenic mutants of ErbB receptors at atomic resolution.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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