Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study.
We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.
Galan
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A
Unidad de Biofisica (Consejo Superior de Investigaciones Cientificas-Universidad del Pais Vasco (CSIC-UPV)) y Departamento de Bioquimica y Biologia Molecular, Universidad del Pais Vasco, Aptdo. 644, 48080 Bilbao, Spain.
Sot
B
B
Llorca
O
O
Carrascosa
J L
JL
Valpuesta
J M
JM
Muga
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
D018835:Chaperonin 10; D018834:Chaperonin 60; D046911:Macromolecular Substances; D021122:Protein Subunits; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D013884:Thiosulfate Sulfurtransferase; D000251:Adenosine Triphosphatases