Mysteries Unravel of Protein-Folding Machine

Could there be a repair mechanism in the human body more remarkable than that of chaperonins, which help misfolded proteins to refold themselves properly? Recent collaborative work by two leading chaperonin researchers has fitted what is known of the structure of a particular such enzyme to a model of its action.1 The result is a wonder to behold. If a protein's amino acid chain fails to fold into its proper or native three-dimensional shape, other proteins can attach to it, creating clumps that threaten the ability of the cell to function. In the bacterium Escherichia coli, a chaperonin called GroEL helps to prevent this. It comprises two large rings, each of which has seven protein subunits that bind adenosine triphosphate (ATP), providing the molecular engine's power. It also is lined with residues that can become hydrophobic, or water-avoiding, enabling binding to a misfolded protein, which leaves some of its own hydrophobic contacts exposed rather than tucked into the folded interior as they should be. ATP binding allows another chaperonin called GroES to attach to GroEL, causing a major conformational change that virtually doubles its size. GroEL assumes the shape of a cage, and the GroES cap fits over it. The misfolded protein falls into the cavity. But before that happens, as GroEL stretches during its conformational change, the attached substrate is likewise stretched. The effect of this stretching force, rather like pulling on a tangled string, will either partially or totally unfold the protein's amino acid chain. The energy that was required to stretch GroEL causes a corresponding loss of ATP's binding energy, and this may play a role in the eventual uncapping of the cage and release of the captive protein. The entire process takes 13 seconds, which is a long time, considering that normal proteins fold in milliseconds. S. Walter Englander, a professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine, suggested in a 1994 paper that the extra time is needed for reorganization of a misfolded molecule.2 George Lorimer, professor of chemistry and biochemistry at the University of Maryland, and colleagues used Englander's theory as a basis of a 1996 "iterative annealing model," which proposed that energy from ATP is coupled to a forceful unfolding of a misfolded protein, followed by its release, and repeated iterations of this process if necessary.3 "Then we got together and decided to do this experiment," Englander recounts. With the help of his student, Mark Shtilerman, the team tracked hydrogen exchange in unfolded Rubisco protein by labeling it with the radioactive hydrogen isotope, tritium. They discovered that the iterative annealing model was correct, that misfolded protein was ejected from the chaperonin regardless of whether or not it had reached its native state. This also disproved another model, which had hypothesized that the substrate remains trapped in the cage until it folds properly. "One doesn't know the exact sequence of events or how the machine functions in detail," Englander observes, "but for the first time, we now have an assay for the activity of this enzyme." He and Lorimer will continue their collaboration. "We'd like to measure the forces that are involved, and we'd like to know how much the protein actually gets unfolded." M. Shtilerman et al., "Chaperonin function: folding by forced unfolding," Science, 284:822-5, April 30, 1999. T.R. Sosnick et al., "The barriers in protein folding," Nature Structural Biology, 1:149-56, 1994. M.J. Todd et al., "Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism," Proceedings of the National Academy of Sciences, 93:4030-5, 1996.

Mysteries Unravel of Protein-Folding Machine

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