Direct observation of protein unfolded state compaction in the presence of macromolecular crowding

Abstract

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

Figure 1

Intramolecular distances displayed on the crystal structure of wild-type S16 (20). The side chain of Trp58 (FRET donor) is colored in green. The dashed lines show the distances between the donor and the two possible acceptor sites (orange). To measure FRET distances, in individual proteins, residues 10 and 74 were changed to cysteines that were subsequently labeled with BODIPY.

Figure 2

Equilibrium unfolding curves for wild-type, W74C, and F10C/W74F in the presence (open circles) and absence (solid circles) of 200 mg/mL dextran. Data are plotted as a function of urea concentration in mol/L (top) and mol/kg H₂O (bottom). The urea concentrations in molar for the dextran samples have been corrected for solvent exclusion effects (see text).

Figure 3

FRET distances fitted as a Gaussian distribution (lines) with an average distance (circles) between the centers of mass of Trp58 and BODIPY for W74C (left) and F10C/W74F (right) in the absence (black lines and circles) and presence (gray lines and circles) of 200 mg/ml dextran at varying concentrations of urea, as indicated in the figure. The areas of the Gaussian distribution have been normalized to unity in all cases.

Figure 4

Tryptophan emission maxima as a function of urea concentration (molal) in the presence (open circles) and absence (solid circles) of 200 mg/mL dextran for W74F and F10C/W74F. The corresponding fraction of unfolding in the presence (gray line) and absence (black line) of 200 mg/mL dextran is displayed in the background.

Figure 5

Far-UV CD spectra of S16 at different molal of urea (0 m (solid line), 2 m (long-dashed line), 8 m (short-dashed line), 10 m (dotted line), and 12 m (dash-dotted line)) with and without dextran for wild-type, W74C, and F10C/W74F.

Figure 6

Chevron plots for folding/unfolding dynamics of wild-type S16 in buffer and in crowding conditions. (Left) Natural logarithms of folding- and unfolding-rate constants are shown as a function of urea for wild-type S16 and F10C/W74F. Solid circles represent buffer conditions and open circles crowded conditions (200 mg/mL dextran). The urea concentrations in the dextran samples have been corrected for solvent exclusion. (Upper left) Data are fitted to Eq. 11. (Lower left) Data points in linear regions are fitted to straight lines, and linear extrapolations of these to 0 M urea were used to determine ln k_f and ln k_u (Table S1). (Right) Change in fluorescence amplitude during each of the kinetic reactions shown at left, reported as the percent change of the initial signal, for wild-type (upper) and F10C/W74F variant (lower).