Yves Bollen, Vrije Universiteit, The Netherlands "Functional protein dynamics measured by NMR spectroscopy and single molecule fluorescence micrsocopy"

"The diffusion of Twin Arginine Translocation complexes in bacterial membranes is substrate and membrane- potential dependent" Siet van den Wildenberg, Felix Oswald, Kah Wai Yau, Holger Lill, Erwin Peterman and Yves Bollen The twin arginine translocation (Tat) system transports fully folded proteins across the inner membrane of bacteria. The energy for translocation is provided by the electrochemical gradient across the inner membrane, called proton motive force. The Tat system is located in the inner membrane and consists of three essential components, TatA, TatB and TatC. TatA is the most abundant Tat protein and is thought to be responsible for pore formation, whereas TatB and TatC together are believed to form a receptor complex, which recognizes and binds to the substrate proteins. We aim to unravel the working mechanism of the Tat system by using fluorescence microscopy. Various components of the system have been fluorescently labelled by genetic fusion to GFP or the red variant mCherry. Fluorescent protein complexes are imaged while they diffuse in the membrane, and data are analyzed in terms of displacement and intensity. Computer simulations are used to better understand the data obtained. We found that the diffusion of the complexes depends on the presence of a substrate and on the membrane potential. "How flavin affects local protein unfolding and associated stability is surprisingly complex" Yves Bollen, Adrie Westphal, Simon Lindhoud, Willem van Berkel and Carlo van Mierlo Binding of cofactors stabilizes proteins against global unfolding. However, how cofactors affect local unfolding and associated stability, and thus protein functionality, is poorly understood. We used H/D exchange followed by NMR to explore local unfolding events in a 179-residue flavodoxin, and were able to pinpoint the residues in the protein that are responsible for its picomolar binding affinity.