Abstract
Hydration properties of folded and unfolded/disordered miniproteins were monitored in frozen solutions by wide-line 1H-NMR. The amount of mobile water as function of T (−80 °C < T < 0 °C) was found characteristically different for folded (TC5b), semi-folded (pH < 3, TCb5(H+)) and disordered (TC5b_N1R) variants. Comparing results of wide-line 1H-NMR and molecular dynamics simulations we found that both the amount of mobile water surrounding proteins in ice, as well as their thaw profiles differs significantly as function of the compactness and conformational heterogeneity of their structure. We found that (i) at around −50 °C ~50 H2Os/protein melt (ii) if the protein is well-folded then this amount of mobile water remains quasi-constant up to −20 °C, (iii) if disordered then the quantity of the lubricating mobile water increases with T in a constant manner up to ~200 H2Os/protein by reaching −20 °C. Especially in the −55 °C ↔ −15 °C temperature range, wide-line 1H-NMR detects the heterogeneity of protein fold, providing the size of the hydration shell surrounding the accessible conformers at a given temperature. Results indicate that freezing of protein solutions proceeds by the gradual selection of the enthalpically most favored states that also minimize the number of bridging waters.
Highlights
Studies aimed at understanding the mechanism of cold denaturing are usually either theoretical, where accessible simulation times render ice-formation unlikely[18,19,20] or www.nature.com/scientificreports/
The applied method allows for quantification of structural heterogeneity (the ratio of heterogeneous binding interface values (HeR)) based directly on the melting diagrams (MD) that report the amount of molten water measured by NMR as a function of temperature[26]
Heating of the frozen sample from −80 °C affords a melting diagram (MD) characteristic for well folded proteins: after the thaw of first hydration layer (T = −53 ± 1 °C, Ea = 4.85 ± 0.02 kJ/mol), a plateau can be seen in the −48 ± 1 °C ≤ T ≤ −26 ± 1 °C temperature range (corresponding to a 5.01 ± 0.02–5.44 ± 0.02 kJ/mol potential energy range Figs 1 and 2S) indicating a temperature region where no additional water molecules melt
Summary
Studies aimed at understanding the mechanism of cold denaturing are usually either theoretical, where accessible simulation times (and the shortcomings of the available water models) render ice-formation unlikely[18,19,20] or www.nature.com/scientificreports/. Net charge +2.5 (>2.5)a +3.5 (>3.5)a experimental, where the application of pressure or additives interferes with ice formation[21,22,23,24] These studies disregard the interaction between the freezing/un-freezing of the hydration layer and of the solute protein molecules. The applied NMR technique relies on the detection and analysis of the limited but significant amount of water that remains mobile around proteins – hydration waters14 – way below 0 °C. This approach allows for the experimental characterization of hindered-rotation barriers and mapping of the energetic heterogeneity of water molecules bound to the molecular surface of proteins[26,27]. Besides being easy to tune, these proteins are small enough to be characterized thoroughly by molecular dynamics simulations (MDSs) which allow, for the first time, enhancement of the NMR results of ice trapped macromolecules with atomistic detail
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