Abstract
We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.
Highlights
It has proven very difficult to directly investigate the thermodynamics and kinetics of native protein folding due to the fact that proteins fold in the cell, often co-translationally
Empigen BB is not a typical denaturant for proteins, and it is important to characterize the interaction of this zwitterionic detergent with myoglobin and heme
As absorbance spectroscopy in the heme region is typically used to quantify myoglobin unfolding [13,42], the absorbance at 409 nm was monitored as a function of detergent concentration (Figure 2A,B)
Summary
It has proven very difficult to directly investigate the thermodynamics and kinetics of native protein folding due to the fact that proteins fold in the cell, often co-translationally. Many natural proteins require the action of chaperones to properly fold into the functional three-dimensional (3D) conformation. The folding process in cells is directly impacted by the incorporation of non-protein cofactors and prosthetic groups such as heme. Only very elegant experimental designs using whole cell extracts and non-native amino acids can begin to address folding in a cellular context [1]. The primary approach to studying protein structural stability has been to use unfolding or denaturation studies using chemical denaturants or thermal energy to unfold the protein. Numerous chemical denaturing agents such as guanidinium chloride, urea, and detergents have been commonly employed to unfold proteins.
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