A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: Application to ribonuclease A

Abstract

A modified pulse-chase experiment is applied to determine if the native-like intermediate I(N) of ribonuclease A is on or off-pathway. The 1H label retained in the native protein is compared when separate samples of 1H-labeled I(N) and unfolded protein are allowed to fold to native in identical conditions. The solvent is 2H2O and the pH* is such that the unfolded protein rapidly exchanges its peptide NH protons with solvent, and I(N) does not. If I(N) is on-pathway, more 1H-label will be retained in the test sample starting with I(N) than in the control sample starting with unfolded protein. The results show that I(N) is a productive (on-pathway) intermediate. Application of the modified pulse-chase experiment to the study of rapidly formed folding intermediates may be possible when a rapid mixing device is used.