Label-free investigation of structure-dynamic relations of single membrane receptors using a tip-enhanced Raman scattering approach
Investigating the dynamics of a single native membrane receptor during a signaling event or in general during its activity requires knowledge about its structure. Obtaining relevant structural information for large molecular systems is still a major challenge. While sensitive detection of products of receptor activities is possible, complex structural changes of the active protein are hardly accessible without labeling. Tip-enhanced Raman scattering (TERS) provides such a route. By combining the electromagnetic field enhancement of a single noble metal nanoparticle mounted onto an AFM cantilever probe and Raman spectroscopy, such a system provides nanometer lateral resolution and at the same time does not require additional labeling that potentially could interfere with physiological function. Based on the size of the particles and the associated signal enhancement even single molecules can be detected. Such a system consequently allows for a direct and label free access to the surface of single membrane receptors.
In the context of this proposal the idea is to investigate the potential of this technique to observe structural changes during a signaling event, ideally by a time trace of consecutive Raman spectra. This way, modifications of the protein sequence or secondary structure that hinder the specific properties can be tracked down with nanometer or even sub-nanometer resolution. The main challenge is the investigation of the general dynamics of receptor system in native membranes under physiological conditions. The plan is to approach the central molecules step by step, starting from fixed isolated active proteins via receptors embedded in model membranes to finally investigate actual cell membranes with single active receptor molecules. In particular the target systems HCN channels, nicotinic acetylcholine receptors and channelrhodopsin-2 will be investigated with respect to their structure-dynamic relationship and the capability of TERS to provide
In two cases the resonance enhancement of the either the receptor itself (e.g. the Schiff base), or the sensitivity towards a resonant molecule (for example fcAMP) will be utilized as a further contrast enhancing mechanism or even as a specific molecular probe. The major task will be establishing a nanometer resolution method for dynamic membrane receptor systems and reliably correlate the information with structural and functional changes of specific single receptor units. Ultimately, by investigating many single membrane receptors separately a better understanding of the averaging effects usually observed in structurally sensitive methods (IR, Raman, NMR etc.) and the nanoscale localization can provide distinct evidence of so far unknown mechanisms.
Volker Deckert will coordinate and supervise the project together with other members of the team.
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