Abstract
Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the (15)N or (13)C' chemical shifts of the unfolded protein at 1 bar to the (1)H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate (15)N shifts differ strongly from natively folded protein, its (13)C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.