Besides, when Hsp70 cannot get a grip on its substrate close to the channel, as would be needed for a power stroke, the motor still works. The researchers engineered a protein with a run of 50 glycine or glutamic acid residues—a stretch that does not bind Hsp70—and showed that it is still imported. Neupert therefore believes that the folded proteins in the cytoplasm “breathe,” and that the unfolded segments of the protein can slide through the channel by thermal fluctuations. Once through the channel, the protein segments are trapped in the mitochondrial matrix by binding to Hsp70. This is similar to the denaturing action of urea, which does not insert itself into folded proteins but binds to temporarily unfolded proteins, thus stabilizing their unfolded structure.
An active motor that pulls is, Neupert admits, “easier to understand. But those more in physical chemistry and equilibria, these people prefer the ratchet.” A ratchet that could tolerate such a long stretch of nonbinding sequence was a bit of a surprise. “There's always the possibility that you are missing something,” says Neupert. “But we think the breathing of the molecule is enough.” ▪