<h3>Title</h3>
<h3>Ionic Liquids and Protein Engineering</h3>
<p><strong><span style="text-decoration: underline;">Abstract</span> :</strong></p>
<p><br /> <strong>Part – I Thermodynamic and Mechanical Stability of Ubiquitin in Ionic Liquids</strong></p>
<p>Biocatalysts are a powerful tool leading the discovery of novel, efficient, non-toxic, and low-cost routes to synthesizing organic compounds. The traditional solvent used for bio-catalysis is water. However, the desired products and precursors are mostly non-polar. Using organic solvents to circumvent this problem results in decreased stability and activity of the enzymes. Additionally, these volatile compounds pose health and environmental hazards. Ionic liquids – low melting temperature solvent consisting of asymmetric, bulky ions, proposes a solution to problems with existing methodology. However, the effect of ionic liquids on protein stability and folding remains to be understood. Moreover, to the best of our knowledge, there are no single-molecule force experimental studies investigating the effect of ionic liquids on the folding of proteins. Here, we take ubiquitin, a well-characterized small protein, to investigate the effect of ionic liquids. Single-molecule and bulk experiments with a variety of ionic liquids: [BMIM][Cl], [bmPyr][Cl], [EMIM][Cl], [BMIM][MeSO4], [EMIM][MeSO4], [BMIM][BF4], [EMIM][BF4], suggest that such non-aqueous (chemical) environment leads to a decrease in the stability of ubiquitin. Furthermore, insights from single-molecule pulling studies validate that those ionic liquids reduce the protein flexibility and decrease the unfolding barrier.</p>
<p><strong> </strong><strong>Part-II Effect of t-SNAREs binding on the mechanics of C2AB domains of Synaptotagmin</strong></p>
<p><strong> </strong> The fusion of cellular membranes is a vital process in different cells, especially during the exocytosis of neurotransmitters at chemical synapses in the brain. The soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 play a crucial role in catalyzing membrane fusion. Besides SNARE proteins, many proteins regulate this process, including Munc13, Munc18, Complexin, Synaptotagmin-1, etc. Synaptotagmin-1 (Syt1) is associated with vesicle, and it acts as a Ca<sup>2+</sup> sensor. It interacts with the SNARE-Complex and negatively charged anionic phospholipids during the Ca<sup>2+</sup> influx and causes fast synchronous exocytosis of synaptic vesicles. However, the exact molecular mechanism by which Syt1 triggers vesicle fusion remains controversial, and many competing models exist. To understand the role of Syt1 in the final step in fusion pore formation, Syt1 has been studied using Single-Molecule Force Spectroscopy by AFM. It was revealed that one of the domains of Syt1 (C2A) unfolds from N-terminal at a relatively small stretching force and can act as a mechanical switch for binding partners of Syt1. However, the major shortcoming of this previous study was the absence of binding partners of Syt1, i.e., t-SNAREs and anionic phospholipids. We have introduced t-SNAREs embedded in phospholipid bilayer nanodisc to mimic biologically relevant conditions. We have planned to do SMFS of C2AB domains in the presence of its binding partners.</p>
<p><strong> </strong><strong>Part-III Revisiting the unconventional linkages in SMFS using non-canonical amino acid protein engineering</strong></p>
<p>Single-molecule force spectroscopy (SMFS) is a versatile tool for studying proteins. It uses force as a more biologically relevant perturbation as compared to the chemical and temperature perturbation. However, a significant shortcoming of most studies of SMFS is the limited direction of stretching, i.e., conventional N-C terminal. These conventional linkages limit the SMFS to explore only the 1-D dimension of a protein's multidimensional potential energy surface. To overcome this, many protein engineering strategies have been implemented, from introducing intermolecular disulfide bonds to recently introducing an internal sortase tag. These strategies have limitations and cannot be generalized for any protein. However, with the rise of new methodology in protein engineering, i.e., incorporating non-canonical amino acids, unconventional linkages in SMFS should be revisited. We have devised strategies for any general protein to form unconventional linkages, including N-terminal to an internal site, C-Terminal to an internal site, two internal sites, and parallel linkages.</p>