The conformational substates BI and BII of the phosphodiester backbone in B-DNA are thought to contribute to DNA flexibility and protein recognition. We have studied by rapid scan FTIR spectroscopy ...the isothermal BI−BII transition on its intrinsic time scale. Correlation analysis of IR absorption changes occurring within seconds after a reversible incremental growth of the DNA hydration shell identifies water populations w1 (PO2 −-bound) and w2 (non-PO2 −-bound) exhibiting weaker and stronger H-bonds, respectively, than those dominating in bulk water. The BII substate is stabilized by w2. The water H-bond imbalance of 3−4 kJ mol−1 is equalized at little enthalpic cost upon formation of a contiguous water network (at 12−14 H2O molecules per DNA phosphate) of reduced ν(OH) bandwidth. In this state, hydration water cooperatively stabilizes the BI conformer via the entropically favored replacement of w2−DNA interactions by additional w2−water contacts, rather than binding to BI-specific hydration sites. Such water rearrangements contribute to the recognition of DNA by indolicidin, an antimicrobial 13-mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the BI conformer in a water-mediated induced fit. The FTIR spectra resolve sequential steps leading from PO2 −-solvation to substate transition and eventually to base stacking changes in the complex. In combination with CD-spectral titrations, the data indicate that, in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the DNA hydration shell allows for entropic contributions similar to those assigned to water upon DNA ligand recognition in solution.
Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis 1. It is assumed that ...sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions 2. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a “dry, yet alive” larva from a “dry and dead” one.
► Caenorhabditis elegans dauer is a true anhydrobiote; it can resist severe desiccation ► Trehalose is essential for desiccation tolerance in C. elegans ► Desiccation damages cellular membranes and membrane-bound organelles ► Trehalose protects membranes by preserving native lipid packing during desiccation
The solvent-solute interface of a biomolecule is a dynamic but yet highly structured domain that links a chemically diverse solute surface to the chemically homogeneous bulk aqueous phase. The role ...of the resulting intermediate domain, i.e. the "hydration shell", in regulating DNA structure and recognition has been addressed here by time-resolved infrared spectroscopy. A highly reproducible automated hydration pulse regime was established and implemented for attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy to monitor the structural response of DNA to an incremental growth of its hydration shell on its intrinsic time scale of seconds. The transition from the crystallographically defined BI to the BII substate of B-DNA was found to be driven by the increase of water disorder upon growth of the hydration shell, derived from the water OH-stretching absorption frequency and band width changes. 2D correlation analysis was used to identify different water clusters from the temporal behaviour of their water OH stretching frequencies. The results show that BII-stabilizing structural constraints are exerted by strong water-DNA H-bonds in the grooves of B-DNA and are relieved when the groove-bound water merges into a contiguous hydration shell with the less H-bonded PO2- -solvation sphere at ~14 water molecules per DNA phosphate. The H-bond imbalance at the disjunct hydration sites is split symmetrically around the average H-bond strength of bulk water. Thus, merging into a contiguous hydration shell proceeds at little enthalpic cost and homogeneous connectivity to the outer bulk-like H-bond network, such that alteration in the network distant from the DNA can regulate the BI-BII transition in a cooperative manner. The water connectivity is disrupted by DNA-binding peptides. Remarkably, the data show that the replacement of hydration shell water upon ligand biding is crucial in conferring substate specific recognition by peptides that have little intrinsic structural preference. The antibacterial peptide indolicidin secreted from bovine neutrophils dehydrates the non-PO2--bound hydration sites, thereby rendering the unstructured peptide highly specific for the BI state with vibrational signature almost identical to the bacterial minor groove binder netropsin. The proposed dominant role of hydration shell water for DNA conformation was challenged by studying the competing effect of structured water in the coordination-shell of the lanthanide Eu3+ on water structure in the DNA hydration shell. Whereas no effect is seen at low hydration, a hydrogen-like phase is formed at a stoichiometric ratio of Eu3+ :DNA:H2O of 1:10:140, characterized by a strong increase of the molar volume of hydration water. This novel phase appears attractive for lanthanide and possibly actine separation approaches based on biomolecular coordination.
The solvent-solute interface of a biomolecule is a dynamic but yet highly structured domain that links a chemically diverse solute surface to the chemically homogeneous bulk aqueous phase. The role ...of the resulting intermediate domain, i.e. the "hydration shell", in regulating DNA structure and recognition has been addressed here by time-resolved infrared spectroscopy. A highly reproducible automated hydration pulse regime was established and implemented for attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy to monitor the structural response of DNA to an incremental growth of its hydration shell on its intrinsic time scale of seconds. The transition from the crystallographically defined BI to the BII substate of B-DNA was found to be driven by the increase of water disorder upon growth of the hydration shell, derived from the water OH-stretching absorption frequency and band width changes. 2D correlation analysis was used to identify different water clusters from the temporal behaviour of their water OH stretching frequencies. The results show that BII-stabilizing structural constraints are exerted by strong water-DNA H-bonds in the grooves of B-DNA and are relieved when the groove-bound water merges into a contiguous hydration shell with the less H-bonded PO2- -solvation sphere at ~14 water molecules per DNA phosphate. The H-bond imbalance at the disjunct hydration sites is split symmetrically around the average H-bond strength of bulk water. Thus, merging into a contiguous hydration shell proceeds at little enthalpic cost and homogeneous connectivity to the outer bulk-like H-bond network, such that alteration in the network distant from the DNA can regulate the BI-BII transition in a cooperative manner. The water connectivity is disrupted by DNA-binding peptides. Remarkably, the data show that the replacement of hydration shell water upon ligand biding is crucial in conferring substate specific recognition by peptides that have little intrinsic structural preference. The antibacterial peptide indolicidin secreted from bovine neutrophils dehydrates the non-PO2--bound hydration sites, thereby rendering the unstructured peptide highly specific for the BI state with vibrational signature almost identical to the bacterial minor groove binder netropsin. The proposed dominant role of hydration shell water for DNA conformation was challenged by studying the competing effect of structured water in the coordination-shell of the lanthanide Eu3+ on water structure in the DNA hydration shell. Whereas no effect is seen at low hydration, a hydrogen-like phase is formed at a stoichiometric ratio of Eu3+ :DNA:H2O of 1:10:140, characterized by a strong increase of the molar volume of hydration water. This novel phase appears attractive for lanthanide and possibly actine separation approaches based on biomolecular coordination.
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