Excess area dependent scaling behavior of nano-sized membrane tethers.

Thermal fluctuations in cell membranes manifest as an excess area ([Formula: see text]) which governs a multitude of physical process at the sub-micron scale. We present a theoretical framework, based on an in silico tether pulling method, which may be used to reliably estimate [Formula: see text] in live cells. We perform our simulations in two different thermodynamic ensembles: (i) the constant projected area and (ii) the constant frame tension ensembles and show the equivalence of our results in the two. The tether forces estimated from our simulations compare well with our experimental measurements for tethers extracted from ruptured GUVs and HeLa cells. We demonstrate the significance and validity of our method by showing that all our calculations performed in the initial tether formation regime (i.e. when the length of the tether is comparable to its radius) along with experiments of tether extraction in 15 different cell types collapse onto two unified scaling relationships mapping tether force, tether radius, bending stiffness κ, and membrane tension σ. We show that [Formula: see text] is an important determinant of the radius of the extracted tether, which is equal to the characteristic length [Formula: see text] for [Formula: see text], and is equal to [Formula: see text] for [Formula: see text]. We also find that the estimated excess area follows a linear scaling behavior that only depends on the true value of [Formula: see text] for the membrane, based on which we propose a self-consistent technique to estimate the range of excess membrane areas in a cell.

Membrane protein stability depends on the concentration of compatible solutes--a single molecule force spectroscopic study.

Compatible solutes are small, uncharged, zwitter ionic, osmotically active molecules produced and accumulated by microorganisms inside their cell to counteract different kinds of environmental stress. They enhance protein stability without interfering with the metabolic pathways even at molar concentrations. In this paper, we report the stabilizing effects of compatible solutes, ectoine, betaine and taurine on membrane protein bacteriorhodopsin at different concentrations. Using atomic force microscopy based single molecule force spectroscopy the impact of the osmolytes was quantified by measuring the forces required to pull the protein out of the membrane and the change in the persistence lengths of the unfolded polypeptide chain. Increase in unfolding forces were observed, indicating the strengthening of intramolecular interactions, which are vital for protein stability. The decrease in persistence lengths was recorded and showed increasing tendencies of the polypeptide strand to coil up. Interestingly, it was revealed that these molecules have different stabilizing effects on protein unfolding at different concentrations. The results show that the unfolding of single protein provides insight to the structure-dynamic relationship between the protein and compatible solute molecules at sub-nanometer scale. This also helps to understand the molecular mechanism involved in protein stabilization by organic osmolytes.

Effect of the compatible solute ectoine on the stability of the membrane proteins.

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.