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.

AFM, DSC and fluorescence spectroscopy have been used to reveal the action mechanism of the antimicrobial peptide (P)GKY20.

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.

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.

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.