Correct protein folding in glycerol

Abstract

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.

Figure 1

The enzymatic activity yield on refolding/reoxidation of hen egg-white lysozyme in glycerol containing small fractions of water and in the pure aqueous solvent. Lysozyme was unfolded and reduced in aqueous solution, refolded and reoxidized in one of the glycerol–water mixtures depicted by the shaded bars, and finally assayed in aqueous solution against dried cells of M. lysodeikticus. For experimental conditions, see Materials and Methods and the legend to Table 1. At each glycerol concentration, the refolding yield (i.e., the recovery of the lysozyme activity) was independently measured at least in quadruplicate; the heights of the shaded bars correspond to the mean values, and the error bars correspond to the standard deviations in these experiments.

Figure 2

The dependence of the recovery of the lysozyme activity upon refolding/reoxidation on the protein concentration during this reaction in aqueous solution (curve a) and in 99% (vol/vol) glycerol (curve b). For experimental conditions, see Materials and Methods and for those in b also the legend to Table 1. Each data point shown was obtained at least in triplicate, and the mean values were plotted with error bars corresponding to the standard deviations from the mean.