title: Folding of an unfolded protein by macromolecular crowding in vitro.
abstract: Protein folding in vivo takes place in a highly crowded environment. The resulting excluded volume forces are thought to stabilize folded forms of proteins. In agreement, many in vitro studies have shown that the presence of macromolecular crowding agents increases the stability of folded proteins but often by only a few kJ per mol. Although it should not matter at what position in the transition between folded and unfolded forms the effect of crowding is employed, there have been no studies assessing whether excluded volume forces alone can correctly fold polypeptides that are mostly unfolded. However, some studies have indicated that the effect of crowding becomes larger the more destabilized the protein is (but still being folded), suggesting that the crowding effect may be exaggerated for unfolded proteins. To address this question directly, we turned to a destabilized mutant of protein L that is mostly unfolded in water but can be folded upon addition of salt. We find that the effect of 200 mg/mL Dextran 20 on the folding equilibrium constant for unfolded protein L (ΔΔGU ≈ 2 kJ mol(-1)) matches the crowding effects found on the folded wild type protein and the mutant when prefolded by salt. This result indicates that the excluded volume effect is independent of starting protein stability and that crowding can shift the reaction toward the folded form when the polypeptide is in the transition region between folded and unfolded states.
authors.affiliation: Department of Chemistry, Umeå University , 90187 Umeå, Sweden.
authors.forename: Jörgen
authors.initials: J
authors.lastname: Adén
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D002942:Circular Dichroism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D046911:Macromolecular Substances