The attenuating ability of deep eutectic solvents towards the carboxylated multiwalled carbon nanotubes induced denatured β-lactoglobulin structure

Abstract

The stabilization of proteins has been a major challenge for their practical utilization in industrial applications. Proteins can easily lose their native conformation in the presence of denaturants, which unfolds the protein structure. Since the introduction of deep eutectic solvents (DESs), there are numerous studies in which DESs act as promising co-solvents that are biocompatible with biomolecules. DESs have emerged as sustainable biocatalytic media and an alternative to conventional organic solvents and ionic liquids (ILs). However, the superiority of DESs over the deleterious influence of denaturants on proteins is often neglected. To address this, we present the counteracting ability of biocompatible DESs, namely, choline chloride–glycerol (DES-1) and choline chloride–urea (DES-2), against the structural changes induced in β-lactoglobulin (Blg) by carboxylated multiwalled carbon nanotubes (CA-MWCNTs). The work is substantiated with various spectroscopic and thermal studies. The spectroscopic results revealed that the fluorescence emission intensity enhances for the protein in DESs. Contrary to this, the emission intensity extremely quenches in the presence of CA-MWCNTs. However, in the mixture of DESs and CA-MWCNTs, there was a slight increase in the fluorescence intensity. Circular dichroism spectral studies reflect the reappearance of the native band that was lost in the presence of CA-MWCNTs, which is a good indicator of the counteraction ability of DESs. Further, thermal fluorescence studies showed that the protein exhibited extremely great thermal stability in both DESs as well as in the mixture of DES-CA-MWCNTs compared to the protein in buffer. This study is also supported by dynamic light scattering and zeta potential measurements; the results reveal that DESs were successfully able to maintain the protein structure. The addition of CA-MWCNTs results in complex formation with the protein, which is indicated by the increased hydrodynamic size of the protein. The presence of DESs in the mixture of CA-MWCNTs and DESs was quite successful in eliminating the negative impact of CA-MWCNTs on protein structural alteration. DES-1 proved to be superior to DES-2 over counteraction against CA-MWCNTs and maintained the native conformation of the protein. Overall, both DESs act as recoiling media for both native and unfolded (denatured by CA-MWCNTs) Blg structures. Both the DESs can be described as potential co-solvents for Blg with increased structural and thermal stability of the protein. To the best of our knowledge, this study for the first time has demonstrated the role of choline-based DESs in the mixture with CA-MWCNTs in the structural transition of Blg. The DESs in the mixture successfully enhance the stability of the protein by reducing the perturbation caused by CA-MWCNTs and then amplifying the advantages of the DESs present in the mixture. Overall, these results might find implications for understanding the role of DES-CA-MWCNT mixtures in protein folding/unfolding and pave a new direction for the development of eco-friendly protein-protective solvents.