Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-5-protein (G5P) to a single-stranded DNA (dT25). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonucleotide, which has important consequences for osmotic regulation mechanisms.

Ectoine is an important osmolyte, which allows microorganisms to survive in extreme environmental salinity. The hygroscopic effects of ectoine in pure water can be explained by a strong water binding behavior whereas a study on the effects of ectoine in salty solution is yet missing. We provide Raman spectroscopic evidence that the influence of ectoine and NaCl are opposing and completely independent of each other. The effect can be explained by the formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics. The mechanism is corroborated by first principles calculations and broadens our understanding of zwitterionic osmolytes in aqueous solution. Our findings allow us to provide a possible explanation for the relatively high osmolyte concentrations in halotolerant bacteria.

Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced freevibrational density of states of water due to ectoine, as well as the use of ectoine as an OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).