Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding.
Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.
Martin
Jörg
J
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Box G-J2, Providence, Rhode Island 02912, USA. joerg.martin@tuebingen.mpg.de
D018835:Chaperonin 10 / Q000737:chemistry / Q000378:metabolism*; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013884:Thiosulfate Sulfurtransferase; D010751:Phosphopyruvate Hydratase