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3. Water is the main controlling factor to retain enzymatic activity inside DES: 
 

To have an idea about the role of water, we measured the solvation dynamics of active-site tagged bromelain.

This is the very first time that water structure modulation of a protein is captured in DES. In all the cases, the relaxation is bi-exponential. The result depicts that the initial decay part, which is mainly controlled by the water in the first solvation shell, is mostly unaltered (at least up to 70% DES), although viscosity changes considerably (see figures 4 and 5). In fact, the change of the slower component is also strongly decoupled from the medium viscosity.

This result is remarkable. It suggests that at least in the first solvation shell, water structure is maintained even at a high concentration of DES, and overall protein feels an aqueous environment even at a very high concentration of DES. Probably, it provides the first experimental proof of why enzymes remain active in DES.

Figure 4. Solvation dynamics of CPM-tagged to bromelain in DES of various hydration. On the left, solvent response; middle variation of the faster time component; and on the right-hand side variation of the slower component of time is shown.

 

 

 

Figure 5. Viscosity measurement of DES with different degrees of hydration

 

We believe this experiment has a huge potential in explaining many things that are previously poorly understood. For example, desiccation-tolerant plants can survive in almost waterless conditions. However, during that, it accumulates osmolytes (sugar and amine acids etc.) that might potentially form DES. It is possible that even in the presence of a very less amount of water, it maintains its structure and dynamics and keeps the proteins active and stable to maintain biophysical processes.

Also, the average solvation time remains somewhat constant with the concentration of DES. Although a huge effort is necessary, this study roughly establishes that maintaining water structure might be one of the most important factors that keep enzymes stable and active in DES.

 

A universal mechanism in the world of complex biophysics:

 

 

 

 

 

 

 

 

 

 

Figure 6. Solvation dynamics of Coumarin 343 in DES of various hydration. On the left, solvent response; middle variation of faster time component; and on the right-hand side variation of slower component of time is shown.

 

As a control experiment, we measured the modulation of water dynamics at various dilutions (with DES) by femtosecond transient absorption spectroscopy. This itself is a vital study as the hydration dynamics of hydrated DES have not been explored in detail. The result is depicted in figure 6. Here also, the solvent correlation function is biexponential in nature. To our delight, the faster component of solvation, which comes from the most nearby water molecules to the solvation probe, remains more or less the same, similar to the result obtained when protein was present. However, the slower component, in this case, roughly follows the bulk viscosity trend. The importance of this result might be enormous. Though biological behaviours are very much system-specific, our result suggests there might be a universal reason for protein stability and activity in DES through associated water modulation, in the world of complex biophysics.

Protein Solvation
viscosity.jpg
TA Solvation
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