Membrane disintegration by the antimicrobial peptide (P)GKY20: lipid segregation and domain formation.

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.

Exploring the effects of cosolutes and crowding on the volumetric and kinetic profile of the conformational dynamics of a poly dA loop DNA hairpin: a single-molecule FRET study.

We investigated the volumetric and kinetic profile of the conformational landscape of a poly dA loop DNA hairpin (Hp) in the presence of salts, osmolytes and crowding media, mimicking the intracellular milieu, using single-molecule FRET methodology. Pressure modulation was applied to explore the volumetric and hydrational characteristics of the free-energy landscape of the DNA Hp, but also because pressure is a stress factor many organisms have to cope with, e.g. in the deep sea where pressures even up to the kbar level are encountered. Urea and pressure synergistically destabilize the closed conformation of the DNA Hp due to a lower molar partial volume in the unfolded state. Conversely, multivalent salts, trimethylamine-N-oxide and Ficoll strongly populate the closed state and counteract deteriorating effects of pressure. Complementary smFRET measurements under immobilized conditions at ambient pressure allowed us to dissect the equilibrium data in terms of folding and unfolding rate constants of the conformational transitions, leading to a deeper understanding of the stabilization mechanisms of the cosolutes. Our results show that the free-energy landscape of the DNA Hp is a rugged one, which is markedly affected by the ionic strength of the solution, by preferential interaction and exclusion of cosolvents as well as by pressure.

Impact of kilobar pressures on ultrafast triazene and thiacyanine photodynamics.

Very short fluorescence lifetimes evidence ultrafast deactivation of photoexcited molecules. To unveil the underlying mechanism for two compounds exhibiting (sub)picosecond emission dynamics, we combine femtosecond fluorescence upconversion with high-pressure liquid-phase spectroscopy. For the triazene berenil, the absence of a pressure dependence corroborates a bicycle-pedal motion as deactivating process. In the thiacyanine NK88 which may undergo a bi-phasic deactivation, our results suggest that kilobar pressures lead to a modification of the excited-state potential energy surface, thereby changing the branching ratio of two competing pathways and opening a possibility to steer the product distribution of the photoreaction.

Pressure and cosolvent modulation of the catalytic activity of amyloid fibrils.

We report on the effects of pressure and cosolvents on the catalytic activity of a designed amyloid fibril by applying a high-pressure stopped-flow methodology with rapid spectroscopic detection. FTIR spectroscopic data revealed a remarkable pressure and temperature stability of the fibrillar catalyst. The activity is further enhanced by osmolytes and macromolecular crowding.

On the Origin of Microtubules' High-Pressure Sensitivity.

For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.

The effects of glycine, TMAO and osmolyte mixtures on the pressure dependent enzymatic activity of α-chymotrypsin.

High pressure is an important feature of certain natural environments, such as the deep sea where pressures up to about 1000 bar are encountered. Further, pressure effects on biosystems are of increasing interest for biotechnological applications, such as baroenzymology. We studied the effect of two different natural osmolyte mixtures, with major components being glycine and trimethylamine-N-oxide (TMAO), on the activity of α-chymotrypsin, using high-pressure stopped-flow methodology in combination with fast UV/Vis detection. We show that pressure is not only able to drastically enhance the catalytic activity and efficiency of the enzyme, but also that glycine has a significant and diverse effect on the enzymatic activity and volumetric properties of the reaction compared to TMAO. The results might not only help to understand the modulation of enzymatic reactions by natural osmolytes, but also elucidate ways to optimize enzymatic processes in biotechnological applications.

Improved activity of α-chymotrypsin on silica particles - A high-pressure stopped-flow study.

Pressure is well known to affect the catalytic rate of enzymes dissolved in solution. To better understand enzyme kinetics at aqueous-solid interfaces, we have carried out a high-pressure stopped-flow activity study of α-chymotrypsin (α-CT) that is adsorbed on silica particles and, for comparison, dissolved in solution. The enzyme reaction was modulated using pressures up to 2000bar and recorded using the high-pressure stopped-flow technique. The results indicate an 8-fold enhancement of the turnover number upon α-CT adsorption and a further increase of the catalytic rate in the pressure range up to 1000bar. From the pressure dependence of the catalytic rate, apparent activation volumes have been determined. In the adsorbed state of α-CT, a pronounced change of the activation volume is found with increasing pressure. Furthermore, owing to suppression of its autolysis, a significantly longer storage time of α-CT can be achieved when the enzyme is adsorbed on silica particles. The results obtained are discussed in terms of a surface-induced selection of conformational substates of the enzyme-substrate complex.

Effect of interfacial properties on the activation volume of adsorbed enzymes.

We have studied the enzymatic activities of α-chymotrypsin (α-CT) and horseradish peroxidase (HRP) that are adsorbed on various chemically modified planar surfaces under aqueous solution. The enzymes were adsorbed on bare quartz, hydrophobic poly(styrene) (PS), positively charged poly(allylamine hydrochloride) (PAH), and negatively charged poly(styrene sulfonate) (PSS). Activation volumes of the enzymes at the aqueous-solid interfaces were determined by using high-pressure total internal reflection fluorescence (TIRF) spectroscopy. Apparently, the pressure response of the adsorbed enzymes strongly depends on the interfacial properties. α-CT can be activated by pressure (increasing enzymatic rate) on negatively charged surfaces like quartz and PSS, whereas HRP is activated by pressure on hydrophobic PS. Corresponding negative activation volumes of -29 mL mol(-1) for α-CT on quartz, -23 mL mol(-1) for α-CT on PSS, and -35 mL mol(-1) for HRP on PS are found. In addition, the absolute activities of α-CT and HRP on quartz, PS, PAH and PSS were determined by UV absorption at ambient pressure. Remarkably, large activities are found on those surfaces that are associated with negative activation volumes. However, Fourier transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode do not indicate major adsorption induced conformational changes of the enzymes at any interface studied. Overall, the results of this study show that the activity of immobilized enzymes can largely be enhanced by the right combination of adsorbent material and applied pressure.

Activation volumes of enzymes adsorbed on silica particles.

The immobilization of enzymes on carrier particles is useful in many biotechnological processes. In this way, enzymes can be separated from the reaction solution by filtering and can be reused in several cycles. On the other hand, there is a series of examples of free enzymes in solution that can be activated by the application of pressure. Thus, a potential loss of enzymatic activity upon immobilization on carrier particles might be compensated by pressure. In this study, we have determined the activation volumes of two enzymes, α-chymotrypsin (α-CT) and horseradish peroxidase (HRP), when they are adsorbed on silica particles and free in solution. The experiments have been carried out using fluorescence assays under pressures up to 2000 bar. In all cases, activation volumes were found to depend on the applied pressure, suggesting different compressions of the enzyme-substrate complex and the transition state. The volume profiles of free and adsorbed HRP are similar. For α-CT, larger activation volumes are found in the adsorbed state. However, up to about 500 bar, the enzymatic reaction of α-CT, which is adsorbed on silica particles, is characterized by a negative activation volume. This observation suggests that application of pressure might indeed be useful to enhance the activity of enzymes on carrier particles.