Why Protein Remain Stable and Active in DES?
The 2022 Noble Prize is awarded to catalysis. It's 4th time in the last 10 years. Even after so much advancement, still, the enzyme remains the most efficient and specific catalyst. However, the problem is, that enzymes/proteins (at least the wild-type ones) are generally stable and active only in water, limiting its use to a huge extent. Francis Arnold tackled this problem by achieving directed evolution through mutation. The mutated proteins remain stable and active in the organic solvents. The work is recognized through the 2018 Nobel Prize.
Another way of tackling the problem could be to find solvent systems, which will be tunable and the enzyme remains stable and active therein. In the last 5 years, there have been many reports demonstrating protein activity and stability in a new kind of solvent, known as deep eutectic solvents (DES). It might be the next game-changer in the field of biocatalysis. However, to date, a logical way forward is missing in this field. One makes a new DES, takes a protein, and studies its stability and activity. However, any research field truly flourishes when physical insights are well understood. During my Ph.D., I will try to gain a physical insight into why protein remains stable and active in DES.
To fulfill this purpose, I will take a combinational approach.
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I will study the activity of enzyme in DES, and it will be correlated with structural and dynamic alteration of the enzyme, and also with modulation of medium properties. These are separately studied, but a correlation might give a new insight.
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The molecular level picture of the medium in terms of structure and dynamics controls its function. For DES, heterogeneity at various lengths and time-scale is identified routinely. I will try to find out what is the impact of such dynamical heterogeneity.
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The fact that water is essential to biology, is almost axiomatic. I will try to contemplate how it decides the enzymatic behavior in DES.
Results:
1. Correlating Enzyme activity with its structure, dynamics, and media’s physical Properties:
Here, we have taken an essential industrial proteolytic plant enzyme bromelain and tried to decipher the behaviour in a ternary DES composed of acetamide, urea, and sorbitol at mole fractions of 0.5, 0.3, and 0.2, respectively (0.5Ac/0.3Ur/0.2Sor), with various degrees of hydration. The chosen DES is non-ionic and liquid at room temperature. This provides us with a unique opportunity to contemplate protein behaviour in a non-ionic DES for the very first time. Our results infer that at a low DES concentration (up to 30% V/V DES), bromelain adopts a more compact structural conformation, whereas, at higher DES concentrations, it becomes somewhat elongated. The microsecond conformational fluctuation time around the active site of bromelain gradually increases with increasing DES concentration, especially beyond 30% V/V. Interestingly, bromelain retains most of its enzymatic activity in the DES, and at some concentrations, the activity is even higher compared with its native state. Furthermore, we correlate the activity of bromelain with its structure, its active-site dynamics, and the physical properties of the medium. Our results demonstrate that the compact structural conformation and flexibility of the active site of bromelain favour its proteolytic activity. Similarly, a medium with increased polarity and decreased viscosity is favourable for its activity. The presented physical insights into how enzymatic activity depends on the protein structure and dynamics and the physical properties of the medium might provide useful guidelines for the rational design of DESs as biocatalytic media.
(see the relative publication here)
Figure: Correlating enzymatic activity with its structure, active-site dynamics and medium physical properties.
2. Multiple Evidences of Molecular Level Heterogeneity in a Non-ionic Biocatalytic Deep Eutectic Solvent:
Deep eutectic solvents (DESs) are new-generation green solvents with exquisite and tuneable properties. Molecular-level heterogeneity has been identified as an intriguing feature of such solvents. Herein, we examined the spatio-temporal heterogeneity of a potential non-ionic biocatalytic DES, acetamide/urea/sorbitol (0.5Ac/0.3Ur/0.2Sor), and compared the result with corresponding binary acetamide/urea (0.6Ac/0.4Ur) DES, and another related non-ionic ternary DES (0.55Ac/0.36Ur/0.09PEG). We tried to understand the effect of the addition of a third component on the spatio-temporal heterogeneity of a DES, and its potential impact on its efficiency as reaction media. The excitation wavelength-dependent emission measurement suggests an introduction of spatial heterogeneity in acetamide/urea/sorbitol compared to spatially homogenous acetamide/urea and acetamide/urea/PEG. The dynamic heterogeneity measurements in terms of solvation dynamics, dielectric relaxation, and rotational and translational diffusion dynamics indicate a length and timescale dependency. Overall, acetamide/urea/sorbitol is dynamically more heterogenous than the other two related DESs. Moreover, an attempt has been made to understand the efficiency of such solvents in terms of viscosity decoupled dynamics. Notably, a decoupling analysis with different time constants of solvation gives us a first-hand hint as to why these solvents might be efficient reaction media, especially in the case of biocatalysis.
Figure: Dynamic heterogeneity (extent of viscosity decoupling) in similar acetamide based non-ionic deep eutectic solvents through various dynamics measurement
(see the relative publication here)
3. Understanding the intricacy of Protein in Deep Eutectic Solvent: Associated water dynamics, conformational fluctuation, and Stability:
This is the very first time that water structure modulation of a protein is captured in DES. We measured the solvation dynamics of human serum albumin (HSA) in the presence of 0.5Ac/0.3Ur/0.2Sor DES. We observed that hydration dynamics is biexponential in nature and the dynamics become slightly slower compared to the huge viscosity change in the media. A careful look at each time component reveals that especially the fast time component is almost unaltered (at least up to 70% v/v DES). 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. Besides the water dynamics, the intrinsic protein dynamics or conformational fluctuation dynamics can also play a role in dictating protein properties. Employing single molecular level FCS, we observed that with the addition of DES, the conformational fluctuation dynamics become progressively slower. Also, at very low concentrations of DES (up to 30%) there is almost no change in the thermal stability of HSA but a constant decrease in stability is observed beyond that concentration. Overall our study reveals that at lower DES concentrations where the structure remains unperturbed and associated water dynamics also do not change much, DES retains the stability of HSA.
Figure: Associated water remains unperturbed with DES addition
(see the relative publication here)
4. Deep Eutectic Solvent Maintains Associated Water Structure of Protein: Potential Implications on Stability and Activity
