Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell

Abstract

We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.

Fig. 1. (A) Refolding yields measured by the recovery of enzymatic activity during refolding of lysozyme in the absence of crowding agents (white bars) and in the presence of 200 g/l Ficoll 70 (black bars) or 100 g/l BSA (grey bars). (B) Initial refolding kinetics measured by the recovery of enzymatic activity during lysozyme refolding in the absence of crowding agents (open squares) and in the presence of 200 g/l Ficoll 70 (filled squares) or 100 g/l BSA (triangles). Refolding yields are normalized relative to those obtained in the absence of crowding agents to show the differences in the kinetics more clearly. The inset shows the complete kinetics.

Fig. 2. Reverse-phase HPLC elution profiles of intermediates observed in the absence (left panels) and presence (right panels) of 200 g/l Ficoll 70, during (A) the initial stages and (B) the later stages of the refolding process. R and N correspond to the fully reduced state and the native state, respectively.

Fig. 3. Analysis of the levels of the reduced protein (R; circles), des-[76-94] (squares) and the native state (N; triangles) during refolding of lysozyme from the fully reduced state. Amounts of protein were determined by measuring peak areas in reverse-phase HPLC chromatograms. The inset shows the early kinetics more clearly. Reduced lysozyme was refolded in the absence (filled symbols) or presence (open symbols) of 200 g/l Ficoll 70. For details see Materials and methods.

Fig. 4. Schematic diagram summarizing the oxidative refolding of lysozyme in the absence and presence of high concentrations of macromolecules. The changes in the rate of the various stages in the refolding process are discussed in the text. Both the reduced protein (R) and the early intermediates (I) are present in solution as an ensemble of species that are relatively unstructured, which facilitates rapid interconversion between them during the disulfide equilibrium phase (Roux et al., 1999; van den Berg et al., 1999c). Subsequent structural collapse from a subset of these early intermediates results in the formation of a limited number of highly native-like three-disulfide intermediates, which appear more rapidly under crowded conditions. Of these, des-[6-127] and des-[64-80] do not accumulate to high levels during refolding since they are converted rapidly to the fully native state. This gives rise to the fast track in lysozyme refolding, the rate of which is increased under crowded conditions. The slow track of lysozyme refolding results from the need for unfolding of the dominant intermediate des-[76-94], and is slower in the presence of crowding. Details of the oxidative refolding of lysozyme have been published (van den Berg et al., 1999c).