Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon.

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus.

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales.

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-9-102 s).