# Diversity of Potential (Bio)Technological Applications of Amino Acid-Based Ionic Liquids
## 2 Bioapplications of AA ILs: An Overview
### Stability and Activity of Enzymes and Proteins
#### 2.5.2 Effect of AA-ILs on the Stability and Structure of Proteins and Enzymes  
Although they show great potential, the effects of AA-based ILs on the stability and structure of proteins and enzymes have been little studied compared to those of second-generation ILs (containing classical cations and anions). So far, we have found that ILs based on charged AA anions and a choline cation, when added to an insulin solution at a 1.5:1 molar ratio, cause a rearrangement of the protein molecule [28]. However, the overall structure remains very close to that of native insulin in solution. Additionally, the thermal stability of the protein was slightly increased in the presence of [Ch]\({}_{2}\)[Asp] and [Ch][Glu] [28]. On the other hand, in a 1% solution of [Ch][Ser], [Ch][Thr], or [Ch][Lys], type I collagen undergoes rearrangement in the polyproline type II helix region, resulting in a more uncoiled and disordered structure [90]. In contrast, 1% [Ch][Phe] causes deformation due to changes in hydrogen bonding, increased order in the protein, or environmental changes around chromophores, resulting in subtle rearrangements in protein conformation. In the presence of any of the four ILs, a slight decrease (1 to 4 \({}^{\circ}\)C) in the thermal stability of collagen is observed [90]. Similarly, Ch-ILs containing charged AA anions such as Arg, Lys, Asp, and Glu induce conformational changes in the _Rapana thomasiana_ hemocyanin (RtH), resulting mainly in an increase in \(\beta\)-structures at the expense of disordered structures [71]. However, the content of \(\alpha\)-helices is preserved, and the molecules appear more ordered. We also found that this modification improves the thermal stability of RtH [71]. The strong effect of the AA anion is also evident from the observed significant changes in the conformation of RtH induced by Emim-AAs [91]. Interestingly, in a medium containing [Emim][Gly], the structure of RtH remains close to that of the native protein; however, this IL significantly stabilizes the protein aggregates that are present in the solution. In contrast, AA anions with longer or branched side chains, such as Leu, Ile, Val, Met, and His, induce the unfolding of RtH, as evidenced by an increase in \(\beta\)-structures, resulting in a slightly more open conformation. Conversely, in a medium containing either [Emim][Trp] or [Emim][Thr], the RtH molecule becomes more compact, the structure is more ordered, and an increase in \(\alpha\)-helices is observed at the expense of \(\beta\)-structures and aggregated structures. All Emim-AAs have a negative effect on the thermal stability of RtH [91]. Interestingly, molecular dynamics (MD) simulations showed that pure [Emim][Trp], [Emim][Ala], and [Emim][Met], as well as their 5% aqueous solutions, efficiently solvated the Trp-cage miniprotein [92]. Compared to protein solutions in water, no structural changes and smaller thermal fluctuations are observed in these six systems, suggesting better thermal stability. These three ILs seem to be promising candidates as co-solvents for the preservation and storage of Trp-cage proteins [92]. At the same time, [Emim][Phe] induces completely reversible conformational changes in horse heart myoglobin, transitioning from \(\beta\)-sheet to helix and back to \(\beta\)-sheet, in up to 200 uM of the IL [93]. It has been shown that the ions bind directly to the protein and change the local viscosity. This not only changes the conformation of myoglobin but also induces self-assembly of the protein molecules, ultimately leading to fibril formation [93]. Regardless of the type of AA anion, all tetramethylguanidine (TMG)-based ILs induce the thermal unfolding of azurin and red fluorescent mCherry protein [94; 95]. The effect is concentration-dependent and stronger for ILs containing hydrophobic AA anions. For example, in the presence of [TMG][Ala], the thermal denaturation temperature of red fluorescent mCherry protein decreased by 23 \({}^{\circ}\)C [95].