Impact of Deep Eutectic Solvents on the Structure and Dynamics of Protein and Enzyme

Author

Anusha Bhattarai, Kennesaw State UniversityFollow

Date of Award

Spring 4-12-2024

Degree Type

Thesis

Degree Name

Master of Science in Chemical Sciences

Department

Department of Chemistry and Biochemistry

Committee Chair/First Advisor

Mohammad Halim

Second Advisor

Chris Dockery

Third Advisor

Bharat Baruah

Abstract

Deep eutectic solvents (DESs), an emerging class of green solvents, which are formed by combination of two or more components (hydrogen bond donor and hydrogen bond acceptor) at a specific composition. These components form a complex network with various intermolecular interactions and show a declining melting point than the main components. DESs are considered biodegradable, sustainable, and non-toxic in nature. Most protein sample preparation and enzyme activation studies are performed in aqueous or organic solvents; however, these solvents mostly unfold the protein/enzyme and disrupt their structure and function. We hypothesize that DES can be used as promising solvents on retaining the folded structure of protein and enhancing the enzyme activation. In this thesis, various non-ionic DES (menthol-acetic acid, and menthol-Thymol) and amino-acid based DES (Proline-Urea) were synthesized. The formation of DESs was confirmed by infrared spectroscopy and principal component analysis (PCA). Differential Scanning Calorimetry (DSC) was used to characterize the thermal properties of DES, where the melting point of DES was detected lower than that of hydrogen bond donor and accepter used to form DESs. An emerging ambient ionization technique DART coupled with mass spectrometry was implemented to detect the stoichiometric combination of DES components. This technique assists to identify various homo-molecular and hetero-molecular clusters in the non-ionic and amino acid-based DESs. The impact of the DES was investigated for two model proteins (lysozyme, α-lactalbumin) and enzyme (trypsin). The protein-DES interactions were monitored by electro spray ionization coupled with LTQ mass spectrometer. Our results showed that non-ionic DESs have modest effect on the folding and unfolding of the protein whereas amino acid-based DES significantly folded and preserved the native structure of protein and enzyme. Moreover, proline-urea DES re-markedly enhanced the trypsin activation compared to other DESs.