@article {2018|2129, title = {Probing the quality control mechanism of the twin-arginine translocase with folding variants of a -designed heme protein.}, journal = {J Biol Chem}, volume = {293}, year = {2018}, month = {2018 05 04}, pages = {6672-6681}, abstract = {

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Tat system to recognize and translocate -designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the trimethylamine--oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme -induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system\&$\#$39;s quality-control mechanism.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Circular Dichroism, Escherichia coli, Escherichia coli Proteins, Heme-Binding Proteins, Hemeproteins, Membrane Transport Proteins, Methylamines, Models, Molecular, Oxidoreductases, N-Demethylating, Periplasm, Protein Folding, Protein Sorting Signals, Protein Stability, Protein Transport, Proton Magnetic Resonance Spectroscopy, Substrate Specificity, Temperature}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000880}, author = {Sutherland, George A and Grayson, Katie J and Adams, Nathan B P and Mermans, Daphne M J and Jones, Alexander S and Robertson, Angus J and Auman, Dirk B and Brindley, Amanda A and Sterpone, Fabio and Tuffery, Pierre and Philippe Derreumaux and Dutton, P Leslie and Robinson, Colin and Hitchcock, Andrew and Hunter, C Neil} } @article {2015|1755, title = {How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory.}, journal = {Proc. Natl. Acad. Sci. Usa}, volume = {112}, year = {2015}, pages = {9270{\textendash}5}, abstract = {

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford-Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard-Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system.

}, keywords = {Atomic Force, Computer Simulation, Hydrophobic and Hydrophilic Interactions, Mechanical, Methylamines, Methylamines: chemistry, Microscopy, Molecular Dynamics Simulation, Normal Distribution, Polymers, Polymers: chemistry, Polystyrenes, Polystyrenes: chemistry, Protein Binding, Protein Conformation, Protein Folding, Proteins, Proteins: chemistry, Software, Solvents, Solvents: chemistry, Stress, Thermodynamics, Urea, Urea: chemistry, Water, Water: chemistry}, isbn = {1215421109}, issn = {1091-6490}, doi = {10.1073/pnas.1511780112}, url = {http://www.pnas.org/content/112/30/9270}, author = {Mondal, Jagannath and Halverson, Duncan and Li, Isaac T S and Guillaume Stirnemann and Walker, Gilbert C and Berne, Bruce J} }