Dynamics of loop 1 of domain I in human serum albumin when dissolved in ionic liquids

We report on the rotational reorientation dynamics associated with loop 1 of domain I within a large multidomain protein (human serum albumin, HSA) when it is dissolved in binary mixtures of ionic liquid (l-butyl-3- methylimidazoliumbis(trifluoromethylsulfonyl)imide ([C₄m][Tf ₂N]), l-butyl-3-methylimidazolium tetrafluoroborate ([C ₄mim] [BF₄]), or l-butyl-3- methylimidazoliumhexafluorophosphate ([C₄mim] [PF₆])) and distilled deionized water (ddH₂O) as a function of temperature and water loading. In IL/2% ddH₂O (v/v) mixtures, loop 1 of domain I is more significantly denatured in comparison to the protein dissolved in aqueous solutions containing strong chemical dénaturants (e.g., 8 M guanidine HCl (Gu·HCl) or urea). As water loading increases, there is evidence for progressive refolding of loop 1 of domain I followed by recoupling with domains I, II, and III in the [C₄mim] [BF₄]/ddH₂O mixtures at 20°C. Above 30% (v/v) water, where domain I appears refolded, the Ac reporter molecule's semiangle steadily decreases from 35° to 20° with increasing water loading. From the perspective of domain I in HSA, this behavior is similar to the effects of dilution from 4 to 0 M Gu·HCl in aqueous solution. Overall, these results lend insight into the tangle of biocatalytic and structural/dynamical mechanisms that enzymes may undergo in ionic liquid-based systems. It will be particularly motivating to extend this work to include enzyme-attuned ionic liquids shown to improve biocatalytic performance beyond that possible in the native (predominantly aqueous) setting.