Structure and Dynamics of Conformational Substates and Intermediates Revealed on Heating from the Glassy States. A Combined Spectroscopic and Calorimetric Approach.

At ambient temperature rapid interconversion of conformers or the lifetime of short-lived intermediates can be in the ms to ps time scale and because of that, characterization is difficult or impossible. These processes are slowed down on cooling towards the glass transition region by many orders of magnitude, and we have utilized this in our project as outlined below. Part (a): In DNA and proteins, dynamics is essential for their biological function, and conformational substates play a decisive role. Rapid transitions between these substates on the ns to ps time scale are a basic problem in ambient temperature studies. Therefore we investigated the structure and dynamics of conformational substates in hydrated DNA on heating from their glassy state and in the glass liquid transition region where transitions between substates are slower by many orders of magnitude. The biologically active form of DNA is B-DNA, and in the literature it is often supposed to consist solely of the so-called BI substate whereas the BII substate had been attributed to a crystal packing artifact. Conclusions of our studies are, first, that the BII substate occurs also in native highly polymeric B-DNA, and that its population increases with decreasing water content until it becomes the dominant substate. These findings suggest that the BI to BII substate interconversion could be a major contributor to the protein recognition process. Second, our studies show that transition between these substates is coupled with migration of water and restructuring of hydration shells. This is consistent with the view that water is an integral part of nucleic acid structure. Third, our studies at low temperatures suggest that B-DNAs biological activity ceases with decreasing temperature once the dynamics of hydration shells is slowed down and because of that, becomes decoupled from substate interconversion. Part (b): Characterization of intermediates occurring in a chemical reaction is the straightforward method for elucidating the mechanism. A good example is carbonic acid, a key compound in biological and geochemical carbonate-containing systems, and conventional textbook wisdom says that it does not exist. We developed a new cryogenic method for isolating and/or characterizing metastable intermediates by sequentially depositing layers of glassy solutions of two reactants, and by inducing reaction on heating, and applied it to isolation and characterization of carbonic acid as intermediate in the protonation of HCO3 - to CO 2 . We further showed that carbonic acid can be sublimated and recondensed without decomposition. Thus, it could be present in comets, on Mars and outer solar system bodies, in interstellar icy grains, and in Earths upper atmosphere. Part (c) Radicals generated by high-energy irradiation of liquid water are one of the basic sources of radiation damage to biomolecules. However, these radicals are short-lived at ambient temperature which often prevents their investigation. We therefore extended our studies of metastable intermediates to that of radicals generated by - irradiation of the various forms of glassy (amorphous) water and of glassy dilute aqueous solutions. These results are important for our understanding of radical formation on -irradiation of the metastable forms of water, of short- lived radicals as one of the basic sources of radiation damage to biomolecules, and of the effects of -irradiation on the solid forms of water in outer space.