From reversible to irreversible: When the membrane nanotube pearling is coupled with phase separation.

Membrane nanotubes, which are ubiquitous in biology and act as channels maintaining transport between different cells and organelles, readily undergo pearling in response to external stimuli. Membrane nanotube pearling involves generation of heterogeneous curvature coupled with redistribution of membrane components that may interfere with the shape recovery of pearled nanotubes. However, the mechanism underlying such delicate process remains unclear and difficult to study at the molecular scale in vivo. By means of molecular dynamics simulation, here we investigate pearling of multi-component membrane nanotubes and reversibility through manipulating system temperature and osmotic pressure. With the equilibrium shape of membrane nanotubes controlled by the osmotic pressure, our results demonstrate that the process of membrane nanotube pearling can be reversible or irreversible, depending on the phase segregation state. For the pearled nanotube releasing high surface energy, different lipid components redistribute along the tube axial direction. Lipids with unsaturated tails prefer gathering at the high-curvature shrinking region, whereas the swelling region is constituted by saturated lipids forming the liquid-ordered phase of a higher bending rigidity. Such curvature sensitive phase segregation minimizes the system free energy by reducing both the membrane bending energy and line tension at the phase boundary. As such, the pearled nanotube fails to recover its shape upon retracting stimuli, suggesting irreversibility of the membrane nanotube pearling coupled with phase separation. Given importance of membrane nanotube pearling in various cellular activities, these results provide a new mechanism of controlling equilibrium shapes of membrane nanotubes in complex cellular environment.

Revealing Topological Barriers against Knot Untying in Thermal and Mechanical Protein Unfolding by Molecular Dynamics Simulations.

The knot is one of the most remarkable topological features identified in an increasing number of proteins with important functions. However, little is known about how the knot is formed during protein folding, and untied or maintained in protein unfolding. By means of all-atom molecular dynamics simulation, here we employ methyltransferase YbeA as the knotted protein model to analyze changes of the knotted conformation coupled with protein unfolding under thermal and mechanical denaturing conditions. Our results show that the trefoil knot in YbeA is occasionally untied via knot loosening rather than sliding under enhanced thermal fluctuations. Through correlating protein unfolding with changes in the knot position and size, several aspects of barriers that jointly suppress knot untying are revealed. In particular, protein unfolding is always prior to knot untying and starts preferentially from separation of two α-helices (α1 and α5), which protect the hydrophobic core consisting of ß-sheets (ß1-ß4) from exposure to water. These ß-sheets form a loop through which α5 is threaded to form the knot. Hydrophobic and hydrogen bonding interactions inside the core stabilize the loop against loosening. In addition, residues at N-terminal of α5 define a rigid turning to impede α5 from sliding out of the loop. Site mutations are designed to specifically eliminate these barriers, and easier knot untying is achieved under the same denaturing conditions. These results provide new molecular level insights into the folding/unfolding of knotted proteins.