Macromolecular crowding remodels the energy landscape of a protein by favoring a more compact unfolded state

Abstract

The interior of cells is highly crowded with macromolecules, which impacts all physiological processes. To explore how macromolecular crowding may influence cellular protein folding, we interrogated the folding landscape of a model beta-rich protein, cellular retinoic acid-binding protein I (CRABP I), in the presence of an inert crowding agent (Ficoll 70). Urea titrations revealed a crowding-induced change in the water-accessible polar amide surface of its denatured state, based on an observed ca. 15% decrease in the change in unfolding free energy with respect to urea concentration (the m-value), and the effect of crowding on the equilibrium stability of CRABP I was less than our experimental error (i.e., < or = 1.2 kcal/mol). Consequently, we directly probed the effect of crowding on the denatured state of CRABP I by measuring side-chain accessibility using iodide quenching of tryptophan fluorescence and chemical modification of cysteines. We observed that the urea-denatured state is more compact under crowded conditions, and the observed extent of reduction of the m-value by crowding agent is fully consistent with the extent of reduction of the accessibility of the Trp and Cys probes, suggesting a random and nonspecific compaction of the unfolded state. The thermodynamic consequences of crowding-induced compaction are discussed. In addition, over a wide range of Ficoll concentration, crowding significantly retarded the unfolding kinetics of CRABP I without influencing the urea dependence of the unfolding rate, arguing for no appreciable change in the nature of the transition state. Our results demonstrate how macromolecular crowding may influence protein folding by effects on both the unfolded state ensemble and unfolding kinetics.

Figure 1

Representative urea denaturation curves of CRABP I in the presence of different amounts of crowding agent at 20 °C (panel A) and 37 °C (panel B); fraction unfolded was determined by monitoring fluorescence at 350 nm. The symbols are the same for both panels: 0 (▲), 75 (□), and 150 (●) g/l Ficoll. The lines through the data are non-linear fits to an equilibrium two-state model.

Figure 2

Representative modified Stern-Volmer plots for unfolded CRABP I at 37 °C in 150 (●) and 0 (○) g/l Ficoll and NATA (in 8.0 M urea) in 150 (▲) and 0 (△) g/l Ficoll. Lines are fits to eq. 1. The inset is the plot of the region near the intercept.

Figure 3

Extent of PEGylation of unfolded CRABP I in 0 or 150 g/l Ficoll at 20 °C. Native protein was used as a control since all three cysteines are inaccessible in the native state in 0 or 150 g/l Ficoll. The number of PEGylated residues is indicated at right.

Figure 4

Unfolding rate of WT* CRABP I at 37 °C plotted as a function of [urea]_eff (A) and of apparent [urea] (B) in 0 (★), 150 (□), 200 (▲), 300 (◆), 350 (▼) g/l Ficoll. Straight-line fits of the data are shown.

Figure 5

Unfolding rate of P85A CRABP I as a function of [urea]_eff at 20 °C (A) and 37 °C (B) in 0 (▲), 75 (□), 150 (●) g/l Ficoll. Straight-line fits of the data are shown.

Figure 6

Crowding favors a more compact denatured state (D), which leads to a smaller difference in free energy between native (N) and D state in crowded solution than one would expect if a more expanded unfolded state (indicated as dashed line) were present in the crowded solution.