Crowded, cell-like environment induces shape changes in aspherical protein

Abstract

How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein-protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region. Our work demonstrates the malleability of "native" proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo.

Fig. 1.

In vitro effects of crowding on VlsE structure and folding. (A) Urea-induced unfolding of VlsE in 0 (filled circles), 50 (open squares), and 100 (open circles) mg/ml Ficoll 70. (B) Semilogarithmic plot of ln k_obs for VlsE unfolding/refolding as a function of urea in the presence of 0 (filled circles) and 100 (open circles) mg/ml Ficoll 70. (C) ln k_obs versus Ficoll 70 (25 mg/ml increments, 0–100 mg/ml) for denaturant jumps to three specific conditions: 0.6 M urea (filled circles), 2.2 M urea (open squares) and 0.9 M urea (filled squares). (D) Far-UV CD spectra of folded (in buffer) and unfolded (in 2 M urea) VlsE (pH 7, 20°C) in 0 (solid trace), 50 (dashed trace) and 100 (dotted trace) mg/ml Ficoll 70. (E) CD spectra of VlsE in 300 mg/ml Ficoll 70 as a function of urea (from 0 to 2.6 M urea). (F) Resulting CD spectra upon refolding attempts of VlsE unfolded in 2 M urea without Ficoll 70 into buffers containing various amounts of Ficoll 70 at >250 mg/ml (indicated in legend).

Fig. 2.

Free-energy landscape for VlsE as a function of T and crowding level. Free-energy diagram as a function of the radius of gyration R_g and the overlap function (χ) for ϕ_c = 0% (bulk), 15%, 25% at various temperatures expressed in k_BT/ε. χ measures the deviation of structural similarity to the crystal (PDB ID code 1L8W); 0 means same as crystal. The color is scaled by k_BT where k_B is the Boltzmann's constant and ε is 0.6 kcal/mol. The native football-shaped species is labeled C (based on 1L8W with the omitted loop inserted), the bean structure is labeled B, the spherical state is named X, and the unfolded state is indicated by U. Note the different x axis scale in C, F, and I versus in A, B, D, E, G, and H.

Fig. 3.

Different nonnative structures induced by crowding and perturbations. (A) Nonnative excess helical contents, H_nn, are shown in red for the in silico bean structure at ϕ_c = 15% and k_BT/ε = 1.13, with the crystal structure as a structural template. (B) A schematic phase diagram of VlsE conformations in the ϕ_c–T (or urea) plane that agrees with both in vitro and in silico data. The solvent-accessible surface area of VlsE is in white and the antigenic IR₆ sequence is in green for all configurations. We use the same annotations as we did in Fig. 2 for the C, B, X, and U states. (C) Surface model of a high-resolution, all-atom reconstruction of the spherical state of VlsE, with the antigenic region IR₆, residues 272–299, highlighted in green. The surface exposure of IR₆ in this reconstructed structure is 41%.