Quench-flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate

Folding of apomyoglobin is characterized by formation of a compact intermed iate that contains substantial helicity. To determine whether this intermed iate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench-flow hydr ogen-exchange pulse labeling techniques with electrospray ionization mass s pectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway cont aining an obligatory intermediate with a significant hydrogen-bonded second ary structure content.