Impact of reconstituted cytosol on protein stability

Abstract

Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.

Fig. 1.

Thermodynamic cycle (cyt, cytosol; dil, dilute).

Fig. 2.

Differences in corresponding to the vertical sides of Fig. 1. Black bars, in reconstituted cytosol (100.0 g dry weight/L); red bars, values in dilute solution. Data are shown for residues observed in both proteins whose values are statistically different from zero.

Fig. 3.

Differences in corresponding to the horizontal sides of Fig. 1. Green bars, for I57A;I37H variant; blue bars, values for I29A;I37H variant. Data are shown for residues observed in both proteins under both conditions whose values are statistically different from zero.

Fig. 4.

Impact of cytosol on stability. The backbone of CI2 (I29A;I37H) is color-coded by in 100.0 g dry weight/L (A) and 130.0 g dry weight/L (B) of reconstituted cytosol [blue, (kcal/mol) > 0.3; cyan, ; orange, ; red, ]. White areas denote prolines and amide protons that either exchanged completely before the second time point or were too broad to observe under one or more conditions. Changes in the average global stability of CI2 under crowded conditions (C). The SDs of the mean of the values for protons that exchange upon global unfolding (41) are indicated by the size of the circle. Cytosol-100 and Cytosol-130 indicate 100.0 g dry weight/L and 130.0 g dry weight/L of the reconstituted cytosol, respectively at pH 6.5, 20 °C. Ficoll (100 g/L, pH 5.4, 37 °C) data are from ref. . PVP (100 g/L, pH 5.4, 37 °C) data are from ref. . BSA (100 g/L, pH 6.5, 20 °C) and lysozyme (100 g/L, pH 6.5, 20 °C) data are from ref. . The lysate, BSA, and lysozyme data were acquired at 20 °C. The stabilizing effects Ficoll and PVP are probably slightly exaggerated because these data were acquired at 37 °C, and crowding-induced stability is predicted to increase with increasing temperature (57).