Bacterial model membranes under the harsh subsurface conditions of Mars.

Biomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment.

Molecular-Scale Liquid Density Fluctuations and Cavity Thermodynamics.

Equilibrium density fluctuations at the molecular level produce cavities in a liquid and can be analyzed to shed light on the statistics of the number of molecules occupying observation volumes of increasing radius. An information theory approach led to the conclusion that these probabilities should follow a Gaussian distribution. Computer simulations confirmed this prediction across various liquid models if the size of the observation volume is not large. The reversible work required to create a cavity and the chance of finding no molecules in a fixed observation volume are directly correlated. The Gaussian formula for the latter probability is scrutinized to derive the changes in enthalpy and entropy, which arise from the cavity creation. The reversible work of cavity creation has a purely entropic origin as a consequence of the solvent-excluded volume effect produced by the inaccessibility of a region of the configurational space. The consequent structural reorganization leads to a perfect compensation of enthalpy and entropy changes. Such results are coherent with those obtained from Lee in his direct statistical mechanical study.

A van der Waals Model of Solvation Thermodynamics.

Exploiting the van der Waals model of liquids, it is possible to derive analytical formulas for the thermodynamic functions governing solvation, the transfer of a solute molecule from a fixed position in the ideal gas phase to a fixed position in the liquid phase. The solvation Gibbs free energy change consists of two contributions: (a) the high number density of all liquids and the repulsive interactions due to the basic fact that each molecule has its own body leading to the need to spend free energy to produce an appropriate cavity to contain the solute molecule; (b) the ubiquitous intermolecular attractive interactions lead to a gain in free energy for switching-on attractions between the solute molecule and neighboring liquid molecules. Also the solvation entropy change consists of two contributions: (a) there is an entropy loss in all liquids because the cavity presence limits the space accessible to liquid molecules during their continuous translations; (b) there is an entropy gain in all liquids, at room temperature, due to the liquid structural reorganization as a response to the perturbation represented by solute addition. The latter entropy contribution is balanced by a corresponding enthalpy term. The scenario that emerged from the van der Waals model is in qualitative agreement with experimental results.

The impact of N-glycosylation on the properties of the antimicrobial peptide LL-III.

The misuse of antibiotics has led to the emergence of drug-resistant pathogens. Antimicrobial peptides (AMPs) may represent valuable alternative to antibiotics; nevertheless, the easy degradation due to environmental stress and proteolytic enzyme action, limits their use. So far, different strategies have been developed to overcome this drawback. Among them, glycosylation of AMPs represents a promising approach. In this work, we synthesized and characterized the N-glycosilated form of the antimicrobial peptide LL-III (g-LL-III). The N-acetylglucosamine (NAG) was covalently linked to the Asn residue and the interaction of g-LL-III with bacterial model membranes, together with its resistance to proteases, were investigated. Glycosylation did not affect the peptide mechanism of action and its biological activity against both bacteria and eukaryotic cells. Interestingly, a higher resistance to the activity of proteolytic enzymes was achieved. The reported results pave the way for the successful application of AMPs in medicine and biotechnological fields.

General Counteraction Exerted by Sugars against Denaturants.

The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water.