Quantitative Evaluation of Myoglobin Unfolding in the Presence of Guanidinium Hydrochloride and Ionic Liquids in Solution

Abstract

The use of ionic liquids in biochemical and biophysical applications has increased dramatically in recent years due to their interesting properties. We report results of a thermodynamic characterization of the chaotrope-induced denaturation of equine myoglobin in two different ionic liquid aqueous environments using a combined absorption/fluorescence spectroscopic approach. Denaturation by guanidinium hydrochloride was monitored by loss of heme absorptivity and limited unfolding structural information was obtained from Förster resonance energy transfer experiments. Results show that myoglobin unfolding is generally unchanged in the presence of ethylmethylimidazolium acetate (EMIAc) in aqueous solution up to 150 mM concentration but is facilitated by butylmethylimidazolium boron tetrafluoride (BMIBF4) in solution. The presence of 150 mM BMIBF4 alone does not induce unfolding but destabilizes the structure as observed by a decrease in threshold denaturant concentration for unfolding and an 80% decrease in the magnitude of ΔGunfolding from 44 kJ/mol in the absence of BMIBF4 to 8 kJ/mol in the presence of 150 mM BMIBF4. Thus, the BMIBF4 significantly destabilizes the myoglobin structure while the EMIAc does not, likely due to differences in anion interaction capabilities. This is confirmed with control studies using NaAc and LiBF4 solutions. EMIAc may be chosen as cosolvent additive with minimal effects on protein structure while BMIBF4 may be used as a supplement in protein folding experiments, potentially allowing access to proteins which have been traditionally difficult to denature as well as designing ionic liquids to match protein characteristics.

Kinetics and mass spectrometric measurements of myoglobin unfolding in aqueous ionic liquid solutions

“…We compare results for four different ILs: 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIBF 4 ); 1-butyl-3-methyl pyrrolidinium tetrafluoroborate (PyrrBF 4 ); 1-ethyl-3methyl imidazolium acetate (EMIAc); and tetramethylguanidinium acetate (TMGAc). In a previous report [7], we quantified myoglobin thermodynamic stabilities in aqueous solutions of BMIBF 4 and EMIAc and showed that BMIBF 4 destabilizes myoglobin while EMIAc has minimal effect on the protein stability.…”

“…The structures and stabilities of proteins in aqueous ionic liquid (IL) solutions have been the focus of numerous recent reports in the literature [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Such studies are motivated by the utilization of ILs in several different biomedical and biochemical techniques [5,[16][17][18][19][20][21], where ILs are present as solutes in aqueous solution.…”

“…Significant data exists to enable design of ILs for many non-biochemical applications [33], but researchers are just beginning to compile comprehensive IL/protein data for prediction and design of biochemical/biomedical applications. A number of groups have studied different ILs series and their effects on protein stability [11,16,[34][35][36], IL-protein surface interactions [9,26,37,38], and protein thermodynamic stability in aqueous IL solutions [5,7]. Low-concentration ILs in aqueous solution often do not denature proteins, but can destabilize their structures as demonstrated by numerous thermodynamic studies [5,7,8,10,15,39].…”

“…A number of groups have studied different ILs series and their effects on protein stability [11,16,[34][35][36], IL-protein surface interactions [9,26,37,38], and protein thermodynamic stability in aqueous IL solutions [5,7]. Low-concentration ILs in aqueous solution often do not denature proteins, but can destabilize their structures as demonstrated by numerous thermodynamic studies [5,7,8,10,15,39]. Several recent reports have focused on IL-protein interactions in -helical proteins and their role in protein destabilization [2,3,8,11,40].…”

“…Several recent reports have focused on IL-protein interactions in -helical proteins and their role in protein destabilization [2,3,8,11,40]. In this context myoglobin, an -helical protein with a heme moiety, has received recent attention in both applied and fundamental studies [7,11,27,39,[41][42][43]. Protein stabilization and/or destabilization by ILs in aqueous solution is often explained with the Hoffmeister anion series [44], but myoglobin may not exactly follow this series [35,36,39].…”