Abstract
The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.
Highlights
Protein folding mediated by a symmetric GroEL-(GroES)2 complex is poorly understood
Protein Folding Can Proceed in Each Ring of the Footballshaped GroEL-GroES Complex—First, we examined how many GFP molecules can fold within the football-shaped complex at the single-molecule level
The fluorescence of GFP molecules was detected at the positions of the GroEL-GroES complexes (Fig. 1B, GFP), indicating that refolded GFP was encapsulated in the GroEL-GroES complexes
Summary
Protein folding mediated by a symmetric GroEL-(GroES) complex is poorly understood. Results: Single-molecule imaging revealed that GFP folding proceeded in both rings of the symmetric GroEL-(GroES) complex. The folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. Because GroES binds alternatively to each ring of GroEL (two-stroke model), an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) exists throughout the reaction cycle, whereas a symmetric GroEL-(GroES) complex (termed the football-shaped complex) is not formed. Our recent studies have shown that the football-shaped and bullet-shaped complexes coexist during the reaction cycle [14], and the formation of the football-shaped complex is promoted by increases in the ATP/ADP ratio [14] and the concentration of denatured protein [15]. Protein Folding in Symmetric GroEL-(GroES) Complexes two-step reaction, and the kinetics are similar to those in the bullet-shaped complex. Our results demonstrate that the same reactions occur in both rings of the football-shaped complex
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