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Johannes Schmid (Med. Univ. Vienna, Vienna, Austria)
Macromolecular complexes are not rigid entities but dynamic "machines", which are in equilibrium with their single components in a process of repetitive association and dissociation. A specific affinity constant gives the number of bound versus free components, but it does not directly give information on the kinetics of dissociation and re-assembly. However, the later feature is often an important determinant of the activity or regulation of a protein complex. We present a technique, which allows measuring the dynamics of complexes in living cells. This could be achieved by combining fluorescence resonance energy transfer (FRET) microscopy, which visualizes close proximities of adjacent fluorophores, with laser-based bleaching of small areas and monitoring the fluorescence recovery after photobleaching (FRAP) - a method, which is usually used to determine the mobility of molecules. Bleaching of a FRET-donor or a FRET-acceptor within a protein complex followed by recording FRAP in three channels, the donor fluorophore, the FRET channel and the acceptor channel, revealed disassembly/re-assembly of a known protein complex as monitored by a single exponential increase of the calculate