We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg∕ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.enzymatic activity | FRET | folding kinetics | thermal denaturation | protein conformational changes P hosphoglycerate kinase (PGK) is a 415-residue metabolic enzyme that produces ATP and is composed of two roughly equally sized subunits connected by a flexible hinge (1). In the crystal structure, the ADP and diphosphoglycerate binding sites, each located at an N and C subunit, are separated. It has been suggested that a large-scale conformational change (2) is necessary to bring the two subunits together when the phosphoryl group is catalytically transferred, and a hinge-bending mechanism has been postulated (3), bringing together both substrates at the inner surfaces of the C and N subdomains (4, 5).It is still unclear how the conformational and folding dynamics of PGK is affected by the interior of a cell, which is heavily crowded by macromolecules (6, 7). Various computational and theoretical studies have been developed to address the effect of volume exclusion exerted by surrounding macromolecules on protein activity inside cells, called the "macromolecular crowding effect" (8). This effect, in addition to weak chemical interactions between proteins and crowders (9), can stabilize the folded states of a protein relative to the unfolded state (10), perturb folding barriers (11,12), and alter folding rates (13) and folding routes (14).Macromolecular crowding could selectively stabilize one folded protein structure over another (8,(15)(16)(17), particularly for proteins that are structurally malleable so their domains aligned in different orientations would have similar free energies (18). Thus, what we regard as the native structur...

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.energy landscape theory ͉ excluded volume effect ͉ Lyme disease ͉ macromolecular crowding ͉ off-lattice model

Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.

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.

How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.