Title
Spectroscopic studies on the comparative refolding of guanidinium hydrochloride denatured hen egg-white lysozyme and Rhizopus niveus lipase assisted by cationic single-chain/gemini surfactants via artificial chaperone protocol
Author
Nuzhat Gull, Mohd Ishtikhar, Sayem Alam, Syedah Noorul Sabah Andrabi, Rizwan Hasan Khan
Year
2017
Journal
RSC Advances
Abstract
Referred to as second generation surfactants, the gemini surfactants have shown promise in various potential areas of surfactant application. Here we report on the comparative refolding of hen egg white lysozyme (HEWL)/Rhizopus niveuslipase (RNL) by cationic gemini (G5, G6)/single-chain surfactant (CTAB) in the artificial chaperone assisted two step method. The studies were carried out in an aqueous medium at a physiological pH of 7.4 using dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy. The results indicate that very small concentrations of gemini surfactants, at which the single-chain homologue was found to be ineffective, refolded the GdCl denatured enzymes. A perusal of DLS data indicates that against the hydrodynamic radius (Rh) of 2.0 ± 0.06 nm/3.5 ± 0.12 nm for the native lysozyme/RNL, the Rh of the enzymes when the refolding was attempted by simple dilution was found to be 5.1 ± 0.16/38.2 ± 0.98 nm. Hydrodynamic radii very near to the native enzyme, i.e., 2.4 ± 0.08 nm/2.2 ± 0.06 nm for lysozyme and 5.2 ± 0.20 nm/4.5 ± 0.22 nm for RNL, were recorded by using 0.005 mM G5/0.005 mM G6 in combination with methyl-β-cyclodextrin. Like dilution the CTAB assisted refolding was also found not to be very impressive and the Rhobserved was far beyond the native value. The CD and fluorescence studies faithfully corroborate with the DLS data. The results obtained from the multi-technique approach are associated with the stronger forces in gemini surfactants owing to the presence of two charged head groups and two hydrocarbon tails. Keeping in view the results, it is strongly suggested that the gemini surfactants assisted artificial chaperone protocol may be effectively used in the refolding of proteins produced in the genetically engineered cells and may also be used in circumventing diseases resulting from protein aggregation/misfolding.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Protein folding, Biochemistry